Function and regulation of adamts-1

ABSTRACT

The present invention relates to ADAMTS-1 and uses thereof. The present invention also relates to fragments of ADAMTS-1 and methods of inhibiting cell growth and metastasis. The present invention also provide methods of identifying inhibitors and activators relating to the function of ADAMTS-1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/709,296, now abandoned, which is a divisional of U.S. patentapplication Ser. No. 11/104,075, filed Apr. 12, 2005 and issued as U.S.Pat. No. 7,696,307, all of which claim priority to U.S. ProvisionalApplication Ser. No. 60/561,429, filed Apr. 12, 2004 and U.S.Provisional Application Ser. No. 60/650,027 filed Feb. 4, 2005 each ofwhich is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support (NIH Grants No.RO1HL074117) and the U.S. Government may therefore have certain rightsin the invention.

BACKGROUND OF THE INVENTION

The members of ADAMTS (A Disintegrin And Metalloproteinase withThromboSpondin motifs) family belong to ADAM (A Disintegrin AndMetalloproteinase) family of multifunctional proteins that display asignificant sequence homology with snake venom metalloproteinases. Theamino-terminal half of ADAMTS is similar to that of ADAM, which containspropeptide, metalloproteinase, disintegrin, and cysteine-rich domains;while the C-terminal half of ADAMTS is completely different and containsthrombospondin type I-like (TSP) motifs that are originally found inthrombospondin 1 and 2 and spacer region. At least 18 members of ADAMTShave been identified. ADAMTS-1 is the first member identified and isexpressed in many embryonic tissues and in tumors. Disruption ofADAMTS-1 gene results in reduced growth, abnormalities in uteral,adrenal, and adipose tissues, and female infertility.

ADAMTS-1 cleaves aggrecan and versican in vitro, however, physiologicsubstrates of ADAMTS-1 remain to be identified. In addition, ADAMTS-1 iscleaved at the spacer region by matrix metalloproteinases (MMPs). Therole of ADAMTS-1 in tumor growth and metastasis is not well established.ADAMTS-1 was found to display anti-angiogenic and anti-tumor activity,however, increased expression of ADAMTS-1 was correlated to the enhancedmetastatic potential of pancreatic cancers, and studies have shown thatADAMTS-1 is one of the genes up-regulated in the breast cancer withelevated metastatic activity.

Thus, there is a need to clarify the biologic role of ADAMTS-1.Furthermore, there is a need to identify compounds and/or compositionsthat can be used to treat cancer or inhibit cell growth.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides isolated polypeptidefragments of ADAMTS-1 that inhibits tumor growth and metastasis.

In some embodiments, the present invention provides compositionscomprising at least two different polypeptide fragment of ADAMTS-1 thatinhibit cell growth and/or metastasis.

In some embodiments, the present invention provides isolatedpolynucleotides encoding a polypeptide fragment of ADAMTS-1 wherein thefragment inhibits metastasis In some embodiments, the present inventionprovides methods for identifying an inhibitor or an activator ofADAMTS-1 cleavage.

In some embodiments, the present invention provides methods foridentifying a heparin inhibitor.

In some embodiments, the present invention provides methods ofidentifying an inhibitor of the metalloproteinase activity of ADAMTS-1.

In some embodiments, the present invention provides methods ofinhibiting metastasis comprising contacting the cell with a polypeptidefragment of ADAMTS-1 that inhibits metastasis and/or a nucleic acid thatencodes a polypeptide fragment of ADAMTS-1 that inhibits cellproliferation or metastasis.

In some embodiments, the present invention provides methods of treatingcancer in an individual comprising administering to the individual atherapeutically effective amount of a polypeptide fragment of ADAMTS-1and/or a nucleic acid that encodes a polypeptide fragment of ADAMTS-1that inhibits cell proliferation or metastasis.

In some embodiments, the present invention provides methods of treatingcancer comprising administering an inhibitor of the metalloproteinaseactivity of ADAMTS-1.

In some embodiments, the present invention provides methods of treatingcancer comprising administering a therapeutically effective amount of acomposition comprising a polypeptide fragment of ADAMTS-1 comprising thespacer/Cys-rich domain or the spacer domain of ADAMTS-1 or a nucleicacid molecule encoding a polypeptide fragment of ADAMTS-1 comprising thespacer/Cys-rich domain or the spacer domain of ADAMTS-1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B depict how ADAMTS-1 undergoes auto-proteolyticcleavage and the self-cleavage of ADAMTS-1 is regulated. FIG. 1A depictsa diagram of the expression constructs. FIG. 1B depicts how cleavage ofADAMTS-1 is blocked by heparin and HS. TA3_(ADAMTS-1) cells werecultured in the absence (lane 1) or presence of 100 μg/ml of heparin(lane 2), HS (lane 3), hyaluronan (lane 4), or CS (lane 5) for 48 hoursand the cell culture supernatants were analyzed by Western blot withanti-v5 antibody.

FIG. 2A through FIG. 2E depict that ADMATS-1 promotes pulmonarymetastasis, while ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) inhibits theprocess. FIG. 2A depicts a representative gross pictures of the mouselungs two-three weeks after i.v. injection of TA3_(wtb) (a-c) orTA3_(ADAMTS-1) (d-f), or TA3_(ADAMTS-1NTCF) (g-i) cells. FIG. 2B depictsthe survival rate of the experimental mice which were injected with theTA3 transfectants intravenously. Total of thirty mice were used for eachtype of transfectants. FIG. 2C depicts that pulmonary metastatic burdenis expressed by weight of the lungs derived from the experimental mice11 days and 18 days after the i.v. injection of the TA3 transfectants.FIG. 2D depicts representative H&E stained lung sections were derivedthe experimental mice injected with TA3_(wtb) (a), TA3_(ADAMTS-1) (b),TA3_(ADAMTS-1NTCF) (c), and TA3_(ADAMTS-1CTCF) (d) cells. Bar, 100 μm.FIG. 2E depicts Western blot analysis of v5-epitope tagged ADAMTS-1protein expressed by TA3_(ADAMTS-1) cells in vivo using anti-v5 mAb. Theproteins were derived from different pulmonary metastases derived fromTA3_(ADAMTS-1) cells. The arrow indicates the mature proteolyticallyactive ADAMST-1, and the arrowhead marks pro-ADAMTS-1.

FIG. 3A through FIG. 3C depict ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)blocks pulmonary metastasis by inhibiting proliferation and survival oftumor cells and by inhibiting tumor angiogenesis. A5-Bromo-2′-deoxy-uridine (Brdu) incorporation assay and in situdetection of apoptotic cells on the sections derived from theexperimental mouse lungs (six days after i.v. injection of TA3transfectants) was performed. Results demonstrated that expression ofADAMTS-1_(NTCF) or ADAMTS-1_(CTCF), but not that of ADAMTS-1_(minusTSP),inhibits proliferation and promotes apoptosis of the tumor cells, andinhibits tumor angiogenesis; while overexpression of full-lengthexogenous ADAMTS-1 on the top of endogenous ADAMTS-1 has weak effect ontumor cell proliferation and apoptosis and promotes tumor angiogenesisin vivo. The quantitative data that reveals the effects of ADAMTS-1 andthe fragments of ADAMTS-1 on tumor cell apoptosis and proliferation andon tumor angiogenesis are shown in FIG. 3B.

FIG. 4A through FIG. 4D depict how ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)block activation of EGFR and ErbB-2 in vivo, while ADAMTS-1 promotesactivation of these receptors and shedding of AR and HB-EGF precursors.FIG. 4A. Tumor cell tracking assay was performed to determine thepulmonary extravasation of TA3 transfectants. 24 hours after i.v.injection of the green fluorescein labeled TA3 transfectants, the micelung were fixed and sectioned. TA3_(wtb) (A-a), TA3_(ADAMTS-1) (A-b),TA3_(ADAMTS-NTCF) (A-c), and TA3_(ADAMTS-1CTCF) (A-d) cells in the lungparenchyma were shown. FIG. 4B. The pulmonary extravasation rates of theTA3 transfectants were expressed as average number of the tumor cellsper microscopic field. FIG. 4C. Activation of EGFR and ErbB-2 in vivo:immunoprecipitation with anti-EGFR(C-a) or anti-ErbB-2 (C-b) antibodywas performed using the protein lysates derived from the mouse lungswhich were implanted without (lanes 1-3) or with TA3_(wtb)(lanes 4-6),TA3_(ADAMTS-1) (lanes 7-9), TA3_(ADAMTS-1NTCF) (lane 10-12), andTA3_(ADAMTS-1CTCF) (lanes 13-15) 24 hours prior. To normalize number ofthe tumor cells that were included in the protein lysates, based on thetumor cell extravasation rates (B), 100 μg of the lung lysates with orwithout TA3_(wtb) cells, 71 μg of the lung lysates containingTA3_(ADAMTS-1) cells, and 143 μg of the lung lysates containingTA3_(ADAMTS-1NTCF) cells, and 130 μg of the lysates containingTA3_(ADAMTS-1CTCF) cells have been used. The precipitated proteins wereanalyzed by Western blotting with anti-phosphotyrosine antibody todetect phosphor-EGFR(C-a, upper panel) or phosphor-ErbB-2 (C-b, upperpanel), or with anti-EGFR(C-a, bottom panel) or anti-ErbB-2 (C-b, bottompanel) antibody to detect total amount of EGFR or ErbB-2, respectively.FIG. 4D. ADAMTS-1 promotes shedding of AR (D-a), HB-EGF (D-b), but notepigen (D-c), and the constitutive shedding of AR and HB-EGF is blockedor inhibited by ADAMTS-1E/Q, respectively (lane 4 in D-a and -b). Cos-7cells were co-transfected with the expression constructs containing cDNAinserts that encode AR, HB-EGF, or epigen precursors without (lane 1) orwith ADAMTS-1_(NTCF) (lane 2), ADAMTS-1_(CTCF) (lane 3), ADAMTS-1E/Q(lane 4), or ADAMTS-1 (lane 5), and the concentrated serum-free culturemedia derived from these co-transfected Cos-7 cells were analyzed usinganti-AR, HB-EGF, or epigen antibody.

FIG. 5A through FIG. 5D depict how the ADAMTS-1 fragments blockactivation of EGFR and ErbB-2; while ADAMTS-1 promotes shedding of ARand HB-EGF. FIG. 5A. Immunoprecipitation with anti EGFR (A-a) oranti-ErbB-2 (A-b) antibody was performed by using the proteins derivedfrom the mouse lungs received TA3_(wtb) (lanes 1-3), TA3ADAMTS-1 (lanes4-6), TA3ADAMTS-1_(minusTSP) (lanes 7-9), TA3ADAMTS-1_(NTCF) (lanes10-12), and TA3ADAMTS-1_(ctcf) (lanes 13-15) intravenously 5 days prior.The precipitated proteins were analyzed Western blotting withanti-phospho-tyrosine antibody to detect phosphor-EGFR (A-a, upperpanel) and phosphor-ErbB-2 (A-b, upper panel), respectively or withanti-EGFR (A-a, bottom panel) or anti-ErbB-2 (A-b, bottom panel)antibody to detect total amount of EGFR or ErbB-2, respectively. FIG.5B. ADAMTS-1 promotes shedding of AR (B-a), HB-EGF (B-b), but not epigen(B-c), and the shedding is blocked by ADAMTS-1E/Q (lane 4). Cos-7 cellswere co-transfected with the expression constructs containing cDNAinserts that encode the EGF family ligand precursors with or without(lane 1) of TA3ADAMTS-1_(NTCF) (lane 2), TA3ADAMTS-1_(CTCF) (lane 3),ADAMTS-1E/Q (lane 4), and ADAMTS-1 (lane 5). FIG. 5C. The cell culturesupernatants derived from the AR-(C-a) or HB-EGF (C-b) transfected Cos-7cells were applied to serum-starved MCF-10A cells without (lane 5-6) orwithout prior absorption of the supernatants with blocking antibodiesagainst AR (a, lane 7-8) or HB-EGF (b, lane 7-8) in the presence of 400ng of ADAMTS-1 (lane 9-10), ADAMTS-1_(NTCF) (lane 11-12), orADAMTS-1_(CTCF) (lane 13-14). Serum free medium alone (lane 1-2) orcontaining 5 ng of AR (a, lane 3-4) or 4 ng of HB-EGF (b, lane 3-4) wasapplied to serum starved MCF-10A cells. Equal amount of the proteinsderived from the MCF-10A cells were analyzed Western blotting withanti-phospho-Erk1/2 to detect phosphor-Erk1/2 or with anti-Erk antibodyto detect total amount of Erk1/2. FIG. 5D. The cleavage fragments ofADAMTS-1 blocks activation of Erk1/2 in HUVECs induced by VEGF 165(D-a), TGF-α (D-c), HB-EGF (D-d), and AR (D-e), but not that induced bybFGF (D-b). HUVECs were applied with SFM alone (lane 1) or containingdifferent GFs alone (lane 2) with 400 ng of ADAMTS-1 (lane 3),ADAMTS-1_(minusTSP) (lane 4), ADAMTS-1_(NTCF) (lane 5) orADAMTS-1_(CTCF) (lane 6). Equal amount of the proteins derived fromHUVECs were analyzed Western blotting with anti-phospho-Erk1/2 to detectphosphor-Erk1/2 or with anti-Erk antibody to detect total amount ofErk1/2.

FIG. 6A through FIG. 6D depict how ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)blocks activation of Erk1/2 kinases in HUVECs induced by AR, HB-EGF, orVEGF₁₆₅. VEGF₁₆₅ (A, 15 ng/ml), bFGF (B, 15 ng/ml), HB-EGF (C, 4 ng/ml),or AR (D, 5 ng/ml) was used alone (lane 2) or in the presence offull-length ADAMTS-1 and ADAMTS-1 fragments (lanes 3-6). Theserum-starved HUVECs were applied with SFM alone (lane 1), or containingdifferent GFs alone (lane 2) or the GFs plus 400 ng of ADAMTS-1 (lane3), ADAMTS-1_(minusTSP)(lane 4), ADAMTS-1_(NTCF) (lane 5) orADAMTS-1_(CTCF) (lane 6). Equal amount of the proteins derived fromthese HUVECs were analyzed Western blotting with anti-phospho-Erk1/2 todetect phosphor-Erk1/2 (upper panels in A-D) or with anti-Erk antibodyto detect total amount of Erk1/2 (bottom panels in A-D).

FIG. 7A through FIG. 7D depict full-length ADMATS-1 promotes pulmonarymetastasis of TA3 cells, while ADAMTS-1E/Q, ADAMTS-1_(NTF), orADAMTS-1_(CTF) inhibits the process. FIG. 7A depicts a schematic diagramof the domain organization of full-length ADAMTS-1 and the differentdeletional and point-mutated ADAMTS-1 that were used in FIGS. 1, 2, 3,and 8. FIG. 7B depicts the expression levels of full-length ADAMTS-1(lane 1), ADAMTS-1E/Q (lane 2), ADAMTS-1_(NTF) (lane 3),ADAMTS-1_(minusTSP-1) (lane 4), or ADAMTS-1_(CTF) (lane 5) by the pooledpopulations of TA3 transfectants. FIG. 7C depicts the survival rates ofthe experimental mice which were injected with the different TA3transfectants intravenously. A total of 12 mice were used for each typeof transfectants. FIG. 7D depicts the pulmonary metastatic burden wasexpressed by the weight of the tumor bearing mouse lungs derived fromthe experimental mice 12 and 20 days after the i.v. injection of the TA3transfectants.

FIG. 8A through FIG. 8C depict the spacer/Cys-rich domain of ADAMTS-1plays a major role in binding of ADAMTS-1 to the ECM and the cells.Western blotting was performed using anti-v5 antibody to determine thedistribution patterns of the v5-epitope tagged full-length ADAMTS-1(lane 1), ADAMTS-1E/Q (lane 2), ADAMTS-1_(NTF+spacer/Cys-rich) (lane 3),ADAMTS-1_(NTF) (lane 4), ADAMTS-1_(minusTSP-1) (lane 5), ADAMTS-1_(CTF)(lane 6), ADAMTS-1_(CTF+spacer) (lane 7), and ADAMTS-1_(3TSP-1) (lane 8)in the cell culture supernatants (FIG. 8A), the ECM materials (FIG. 8B),and the EDTA-lifted Cos-7 cells (FIG. 8C) that were transfected with thecorresponding expression constructs.

FIG. 9A through FIG. 9D depict how ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)blocks activation of Erk1/2 kinases in HUVECs induce by AR, HB-EGF, orVEGF₁₆₅. VEGF₁₆₅ (A, 15 ng/ml), bFGF (B, 15 ng/ml), HB-EGF (C, 4 ng/ml),or AR (D, 5 ng/ml) was used alone (lane 2) or in the presence offull-length ADAMTS-1 and ADAMTS-1 fragments (lanes 3-6). Theserum-starved HUVECs were applied with SFM alone (lane 1), or containingdifferent GFs alone (lane 2) or the GFs plus 400 ng of ADAMTS-1 (lane3), ADAMTS-1_(minusTSP)(lane 4), ADAMTS-1_(NTCF) (lane 5) orADAMTS-1_(CTCF) (lane 6). Equal amount of the proteins derived fromthese HUVECs were analyzed Western blotting with anti-phospho-Erk1/2 todetect phosphor-Erk1/2 (upper panels in A-D) or with anti-Erk antibodyto detect total amount of Erk1/2 (bottom panels in A-D).

FIG. 10 depicts expression of ADAMTS-1. Expression of ADAMTS-1 wasassessed by RT-PCR using RNAs derived from TA3wt, TA3_(wt1), Lewis lungcarcinoma cells, CMT-93 colon carcinoma cells, B 16F1 and F10 cells, 3T3fibroblasts, C₂C₁₂ myoblasts, and mouse placenta (lanes 2-10).Expression of β-actin by these cells was used as controls. In lane 1,reverse transcriptase was not included in RT reaction with RNA derivedfrom TA3_(wt1) cells.

FIG. 11A and FIG. 11B depict how ADAMTS-1 promotes tumor growth whilethe cleavage fragments of ADAMTS-1 inhibit tumor growth. Growth rates ofthe s.c. tumors derived from different TA3 transfectants are expressedas the means of tumors volumes +/−SD. Total of fifteen mice were usedfor each type of transfectants.

FIG. 12A and FIG. 12B depict the cleavage fragments of ADAMTS-1 blockssubcutaneous tumor growth by inhibiting proliferation and survival oftumor cells, and inhibiting tumor angiogenesis in vivo. The s.c. tumorswere section 12 days after implanting TA3_(wtb) (A, a-d), TA3ADAMTS-1(A, e-h), TA3ADAMTS-1_(NTCF) (A, i-l), and TA3ADAMTS-1_(CTCF) (A, m-p).These sections were stained with H&E (A-a, e, I, m), or reacted withApoptag to detect apoptotic tumor cells in situ (A-b, f, j, n),anti-Brdu antibody to detect proliferating tumor cells (A-c, g, k, o),or with anti-vWF antibody to reveal blood vessels with the tumors (a-d,h, l, p). Bar: 120 μm. The quantitative data that reveals the effects ofADAMTS-1 and the fragments of ADAMTS-1 on tumor cell apoptosis andproliferation in vivo and on tumor angiogenesis are shown in panels B-a,b, c, respectively.

FIG. 13A through FIG. 13D depicts ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)blocks activation of Erk1/2 kinases in HUVECs induce by AR, HB-EGF, orVEGF₁₆₅. VEGF₁₆₅ (A, 15 ng/ml), bFGF (B, 15 ng/ml), HB-EGF (C, 4 ng/ml),or AR (D, 5 ng/ml) was used alone (lane 2) or in the presence offull-length ADAMTS-1 and ADAMTS-1 fragments (lanes 3-6). Theserum-starved HUVECs were applied with SFM alone (lane 1), or containingdifferent GFs alone (lane 2) or the GFs plus 400 ng of ADAMTS-1 (lane3), ADAMTS-1_(minusTSP) (lane 4), ADAMTS-1_(NTCF) (lane 5) orADAMTS-1_(CTCF) (lane 6). Equal amount of the proteins derived fromthese HUVECs were analyzed Western blotting with anti-phospho-Erk1/2 todetect phosphor-Erk1/2 (upper panels in A-D) or with anti-Erk antibodyto detect total amount of Erk1/2 (bottom panels in A-D). The ADAMTS-1fragments block activation of EGFR and ErbB-2; while ADAMTS-1 promotesshedding of AR and HB-EGF. A. Immunoprecipitation with anti EGFR (A-a)or anti-ErbB-2 (A-b) antibody was performed by using the proteinsderived from the mouse lungs received TA3_(wtb) (lanes 1-3), TA3ADAMTS-1(lanes 4-6), TA3ADAMTS-1_(minusTSP) (lanes 7-9), TA3ADAMTS-1_(NTCF)(lanes 10-12), and TA3ADAMTS-1_(ctcf) (lanes 13-15) intravenously 5 daysprior. The precipitated proteins were analyzed Western blotting withanti-phospho-tyrosine antibody to detect phosphor-EGFR (A-a, upperpanel) and phosphor-ErbB-2 (A-b, upper panel), respectively or withanti-EGFR (A-a, bottom panel) or anti-ErbB-2 (A-b, bottom panel)antibody to detect total amount of EGFR or ErbB-2, respectively. B.ADAMTS-1 promotes shedding of AR (B-a), HB-EGF (B-b), but not epigen(B-c), and the shedding is blocked by ADAMTS-1E/Q (lane 4). Cos-7 cellswere co-transfected with the expression constructs containing cDNAinserts that encode the EGF family ligand precursors with or without(lane 1) of TA3ADAMTS-1_(NTCF) (lane 2), TA3ADAMTS-1_(CTCF) (lane 3),ADAMTS-1E/Q (lane 4), and ADAMTS-1 (lane 5). C. The cell culturesupernatants derived from the AR-(C-a) or HB-EGF (C-b) transfected Cos-7cells were applied to serum-starved MCF-10A cells without (lane 5-6) orwithout prior absorption of the supernatants with blocking antibodiesagainst AR (a, lane 7-8) or HB-EGF (b, lane 7-8) in the presence of 400ng of ADAMTS-1 (lane 9-10), ADAMTS-1_(NTCF) (lane 11-12), orADAMTS-1_(CTCF) (lane 13-14). Serum free medium alone (lane 1-2) orcontaining 5 ng of AR (a, lane 3-4) or 4 ng of HB-EGF (b, lane 3-4) wasapplied to serum starved MCF-10A cells. Equal amount of the proteinsderived from the MCF-10A cells were analyzed Western blotting withanti-phospho-Erk1/2 to detect phosphor-Erk1/2 or with anti-Erk antibodyto detect total amount of Erk1/2. D. The cleavage fragments of ADAMTS-1blocks activation of Erk1/2 in HUVECs induced by VEGF 165 (D-a), TGF-α(D-c), HB-EGF (D-d), and AR (D-e), but not that induced by bFGF (D-b).HUVECs were applied with SFM alone (lane 1) or containing different GFsalone (lane 2) with 400 ng of ADAMTS-1 (lane 3), ADAMTS-1_(minusTSP)(lane 4), ADAMTS-1_(NTCF) (lane 5) or ADAMTS-1_(CTCF) (lane 6). Equalamount of the proteins derived from HUVECs were analyzed Westernblotting with anti-phospho-Erk1/2 to detect phosphor-Erk1/2 or withanti-Erk antibody to detect total amount of Erk1/2.

FIG. 14 depicts the domain organization of ADAMTS-1. The various domainsof ADAMTS-1 are shown.

FIG. 15 depicts possible mechanisms of ADAMTS-1 function. 1) Full-lengthADAMTS-1 promotes tumor growth and metastasis by enhancing tumor cellproliferation/survival and tumor angiogenesis throughshedding/activating HB-EGF and AR transmembrane precursors and bypromoting tumor cell invasion through degrading versican; 2) full-lengthADAMTS-1 binds to their substrates through its spacer/Cys-rich domaindirectly or indirectly through binding to HSPGs. Thus, the whole ordifferent parts of the spacer/Cys-rich domain can be used as a dominantnegative regulator of the full-length ADAMTS-1 (by regulating thesubstrate-binding of ADAMTS-1) and to regulate its own cleavage status(to promote proteolytic cleavage of ADAMTS-1, therefore generateanti-tumor fragments); 3) the anti-tumor activity of the ADAMTS-1fragments resides in the TSP-1 domains, which exerts the anti-tumoractivity by inhibiting bioactivity of several soluble heparin bindinggrowth/angiogenic factors including AR and HB-EGF. Thus, the whole orparts of ADAMTS-1NTF (ADANTS-1NTFE/Q) and/or ADAMTS-1CTF can be used toinhibit cancers.

FIG. 16A through FIG. 16D depict full-length ADAMTS-1 and the ADAMTS-1fragments displayed opposite effects on growth and metastasis of LLCcells. FIG. 16A. The expression level of the v5-epitope tagged ADAMTS-1(lane 1), ADAMTS-1E/Q (lane 2), ADAMTS-1_(NTF) (lane 3),ADAMTS-1_(minusTSP-1) (lane 4), ADAMTS-1_(CTF) (lane 5),thrombospondin-1 (lane 6) and thrombospondin-2 (lane 7) by the pooledLLC transfectants. FIG. 16B. The growth rates of the s.c. tumors derivedfrom the different LLC transfectants are expressed as the means oftumors volumes +/−SD. A total of 15 mice were used for each type oftransfectants. FIG. 16C. Survival rates of the experimental mice afterremoval of the s.c. tumors derived from the different LLC transfectants.A total of thirty mice were used for each type of transfectants. FIG.16D. Pulmonary metastatic burden is expressed by the average weight ofthe lungs derived from experimental mice three weeks after removal ofthe s.c. tumors.

FIG. 17A through FIG. 17C depict the metalloproteinase activity ofADAMTS-1_(NTF) is not required for its anti-tumor activity. FIG. 17A.The expression level of the v5-epitope tagged ADAMTS-1_(NTF) (lane 1-3)and ADAMTS-1_(NTF)E/Q(lane 4-6) by the TA3 transfectants. FIG. 17BSurvival rates of the experimental mice after i.v. injection of1×10⁶/mouse TA3 transfectants. A total of 15 mice were used for eachtype of transfectants. FIG. 17C. Pulmonary metastatic burden isexpressed by the average weight of the lungs derived from theexperimental mice three weeks after the iv injection.

FIG. 18 depicts the multiple amino acid sequence alignment of the secondand third repeats of thrombospondin-1 and _(m and c)TSP-1 domains inADAMTS-1, and the deletional and peptide generation strategy in theTSP-1 domains of ADAMTS-1. The deletions and generation of threedifferent peptides s in m and cTSP-1 domains of ADAMTS-1 are shown.

DETAILED DESCRIPTION

In the present invention it has been discovered that ADAMTS-1 isexpressed by many tumor cells and overexpression of ADAMTS-1 promotesgrowth and metastasis of TA3 mammary carcinoma cells by promotingsurvival, proliferation, invasiveness of the tumor cells and tumorangiogenesis in vivo. Additionally, disclosed herein is that ADAMTS-1undergoes auto-proteolytic cleavage to generate N- and C-terminalcleavage fragments that contain at least one TSP type I motif.Auto-proteolytic cleavage of ADAMTS-1 is blocked by heparin and heparinsulfate (HS). Although not bound by any theory, this indicates that theself-cleavage is regulated by HS and heparin sulfate proteoglycans(HSPGs). Thus, as described herein, ADAMTS-1 expressed by TA3 cells ismaintained in the full-length form in vivo to exert pro-tumor growth andmetastasis activity. In contrast to the full-length ADAMTS-1,overexpression of the N- or C-terminal fragment of ADAMTS-1(ADAMTS-1_(NTCF) and/or ADAMTS-1_(CTCF)) inhibits subcutaneous (s.c.)growth of TA3 cells and blocks pulmonary metastasis of the cells byinhibiting proliferation and inducing apoptosis of the tumor cells andby inhibiting tumor angiogenesis. Additionally, the anti-tumor effect ofthe ADAMTS-1 fragments requires a TSP type-I motif. The direct evidencewas provided for the first time that ADAMTS-1 promotes tumor growth andmetastasis, and can serve as a target for cancer therapy.

For the first time, it has been demonstrated that unlike full-lengthADAMTS-1 which promotes shedding of the EGF family ligands includingamphiregulin (AR) and heparin-binding EGF (HB-EGF) and activation of EGFreceptor (EGFR) and ErbB-2, the cleavage fragments of ADAMTS-1 inhibitsactivation of EGFR and ErbB-2 in vivo, and interferes with Erk1/2kinases activation induced by soluble AR. HB-EGF, and/or VEGF in mammaryepithelial cells and endothelial cells. These different effects likelyunderlie the opposite roles of ADAMTS-1 and its cleavage fragments intumor growth and metastasis, suggesting the ADAMTS-1 fragments and theinhibitors of ADAMTS-1 can be most successfully used to treat thecancers overexpressing these heparin binding growth and angiogenicfactors and with activated erbB-signaling pathways.

The term “ADAMTS-1_(NTCF)” can also be referred to as “ADAMTS-1_(NTF)”.The term “ADAMTS-1_(CTCF)” can also be referred to as “ADAMTS-1_(CTF)”.In some embodiments, ADAMTS-1_(NTCF) comprises SEQ ID NO: 9 and/or 11.In some embodiments, ADAMTS-1_(CTCF) comprises SEQ ID NO: 5 and/or 7.

The discovery that ADAMTS-1 can be cleaved into at least two fragmentshas led to the following invention. In some embodiments, the presentinvention provides an isolated polypeptide comprising a fragment ofADAMTS-1 that inhibits cell growth or cell survival and/or metastasis.

As used herein, the term “isolated polypeptide fragment” refers to apolypeptide fragment that is free of the full length protein. In someembodiments, the isolated polypeptide is also free of nucleic acidmolecules. In some embodiments, the isolated polypeptide is free ofcellular membranes. In some embodiments, the isolated polypeptide hasbeen purified away from cellular components. In some embodiments, thepolypeptide comprises a fragment of SEQ ID NO: 1 and/or SEQ ID NO: 3. Insome embodiments, the fragment of ADAMTS-1 comprises SEQ ID NO: 5, 7, 9,and/or 11. The fragment of ADAMTS-1 can be any length such that it isnot the full-length ADAMTS-1 protein. In some embodiments, the fragmentcomprises about 100 to about 150, about 100 to about 200, about 100 toabout 300, about 100 to about 400, about 100 to about 500, about 100 toabout 600, about 100 to about 700, about 100 to about 800, about 100 toabout 900, or about 100 to 950 amino acid residues. In some embodiments,the fragments of ADAMTS-1 comprise modifications of the polypeptidesequence. The modification can be any modification including, but notlimited to, mutations, insertions, substitutions, deletions, and thelike. In some embodiments, the fragment comprises a mutation of Glu toGln. In some embodiments, the mutation of Glu to Gln occurs at aposition corresponding to position 386 (in mouse ADAMTS-1) in the fulllength protein. One of skill in the art can determine what position in afragment corresponds to position 386 in the full length protein (e.g.position 385 in human ADAMTS-1). One of skill in the art can do this by,for example, performing an alignment using any alignment software orBLAST software using default settings. Examples of software that can beused include, but are not limited to, BLAST, GCG, and MacVector™. Insome embodiments, the polypeptide fragment containing a mutationcomprises SEQ ID NO: 33 and/or 35 or a nucleic acid molecule encodingthe same. In some embodiments, the nucleic acid molecule encoding thefragment comprises SEQ ID NOs: 34 and/or 36.

In some embodiments the fragments of ADAMTS-1 are linked to anon-ADAMTS-1 molecule. In some embodiments, the non-ADAMTS-1 molecule isa toxin, peptide, polypeptide, small molecule, drug, and the like. Insome embodiments, the non-ADAMTS-1 molecule is a 6-His-tag, GSTpolypeptide, HA tag, the Fc fragment of human IgG and the like. In someembodiments, the proteinase cleavage sites will be put before the tagsequences, so that after purification these tags can be removed byproteolytic cleavage. For example, the HRV 3C (human rhinovirus type 143C) protease cleavage site (LEVLFQ↓GP-SEQ ID NO:46) can be locatedbefore the COOH-terminal v5 and His epitope tags. The HRV 3C proteasespecifically claves the sequence LEVLFQ↓GP at 40C and were used toefficiently removal the COOH-terminal tags (Novagen).

In some embodiments, the fragment of ADAMTS-1 is fused to anotherpolypeptide that is derived from a protein that is not ADAMTS-1. In someembodiments two fragments from ADAMTS-1 are fused or linked together. Insome embodiments, the two fragments are identical. In some embodiments,the fragments are different from one another. The fragments that can belinked or fused together are ADAMTS-1_(CTCF) (SEQ ID NO: 5 and/or SEQ IDNO:7), ADAMTS-1_(NTCF) (SEQ ID NO: 9 and/or 11), and ADAMTS-1_(spacer)or ADAMTS-_(1spacer/Cys-rich) to achieve maximal anti-tumor efficiency,however any two fragments from ADAMTS-1 can be fused together.

In some embodiments, the present invention provides nucleic acidmolecules encoding a fragments of ADAMTS-1. In some embodiments, thefragments of ADAMTS-1 that inhibits cell proliferation or metastasiscomprise a TSP type-I motif.

A fragment that inhibits cell proliferation or metastasis can also bereferred to as a fragment that inhibits cancer or a fragment can be usedto treat cancer.

As used herein, the term “inhibit cell proliferation” refers to anymeasurement of cell proliferation. A fragment, compound, or compositionthat causes a cell to undergo necrosis or apoptosis is considered toinhibit cell proliferation. Cell proliferation can also be referred toas cell growth or cell division.

Methods of measuring cell proliferation, division, and metastasis areroutine and any method can be used.

For example, one can measure cell invasion using Matrigel in vitro.Metastasis can also be measured and/or observed in vivo by injecting amouse with a tumor cell and determining if the cell spreads to adifferent location away from the sight of injection. Metastasis can alsobe measured by measuring or observing tumor burden or tumor growth inareas that are distinct from the primary tumor location. Cellproliferation can be measured, for example, by counting cells. Celldivision can be measured, for example, by monitoring what phase of thecell cycle a cell or a population of cells is in by using flow cytometryor FACS. Determining if a cell or cell population is dividing isroutine.

In some embodiments, the present invention provides a fragment ofADAMTS-1 that lacks a TSP motif. In some embodiments, the presentinvention provides a deletion of ADAMTS-1 that lacks a TSP motif. Insome embodiments, a polypeptide of ADAMTS-1 that lacks a TSP motifcomprises SEQ ID NO:13 and/or SEQ ID NO:15. The term“ADAMTS-1_(minus TSP)” can also be referred to as“ADAMTS-1_(minus TSP-1)”. In some embodiments, the present inventionprovides a nucleic acid molecule that encodes for a ADAMTS-1 polypeptidethat lacks a TSP motif. In some embodiments the nucleic acid molecule isisolated. In some embodiments the nucleic acid molecule comprises SEQ IDNO: 14 and/or SEQ ID NO: 16.

In some embodiments, the present invention provides an isolated nucleicacid molecule (polynucleotide) encoding a polypeptide fragment ofADAMTS-1.

As used herein the term “isolated nucleic acid molecule encoding apolypeptide fragment of ADAMTS-1” refers to a nucleic acid molecule isfree of a nucleic acid molecule encoding full length ADAMTS-1.

In some embodiments, a fragment encoded by the nucleic acid molecule caninhibit cell proliferation and/or metastasis. In some embodiments, thenucleic acid molecule comprises a fragment of a nucleic acid moleculeencoding a polypeptide comprising SEQ ID NO:1 and/or SEQ ID NO: 2. Insome embodiments the nucleic acid molecule comprises a fragment of SEQID NO: 3 and/or SEQ ID NO: 4 In some embodiments, the nucleic acidmolecule encodes a polypeptide comprising SEQ ID NOs: 5, 7, 9, and/or11. In some embodiments, the nucleic acid molecule comprises SEQ ID NOs:6, 8, 10, and/or 12. In some embodiments, the nucleic acid moleculeencoding a fragment of ADAMTS-1 is operably linked to a promoter. Insome embodiments, the promoter can facilitate the expression in aprokaryotic cell and/or eukaryotic cell. The promoter can be anypromoter that can drive the expression of the nucleic acid molecule.Examples of promoters include, but are not limited to, CMV, SV40, pEF,actin promoter, and the like. In some embodiments, the nucleic acidmolecule is DNA or RNA. In some embodiments, the nucleic acid moleculeis a virus, vector, or plasmid. In some embodiments, the expression ofthe nucleic acid molecule is regulated such that it can be turned on oroff based on the presence or absence of a regulatory substance. Examplesof such a system include, but is not limited to a tetracycline-ON/OFFsystem.

In some embodiments, the nucleic acid molecule is a recombinant viralvector. “A recombinant viral vector” refers to a construct, based uponthe genome of a virus, that can be used as a vehicle for the delivery ofnucleic acids encoding proteins, polypeptides, or peptides of interest.Recombinant viral vectors are well known in the art and are widelyreported. Recombinant viral vectors include, but are not limited to,retroviral vectors, adenovirus vectors, adeno-associated virus vectors,and lenti-virus vectors, which are prepared using routine methods andstarting materials.

Using standard techniques and readily available starting materials, anucleic acid molecule may be prepared. The nucleic acid molecule may beincorporated into an expression vector which is then incorporated into ahost cell. Host cells for use in well known recombinant expressionsystems for production of proteins are well known and readily available.Examples of host cells include bacteria cells such as E. coli, yeastcells such as S. cerevisiae, insect cells such as S. frugiperda,non-human mammalian tissue culture cells Chinese hamster ovary (CHO)cells or Cos-7 cells, and human tissue culture cells such as 293 cellsor HeLa cells.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert DNA molecules into acommercially available expression vector for use in well knownexpression systems. For example, the commercially available plasmidpSE420 (Invitrogen, San Diego, Calif.) may be used for production ofimmunomodulating proteins in E. coli. The commercially available plasmidpYES2 (Invitrogen, San Diego, Calif.) may, for example, be used forproduction in S. cerevisiae strains of yeast. The commercially availableMAXBAC™ complete baculovirus expression system (Invitrogen, San Diego,Calif.) may, for example, be used for production in insect cells. Thecommercially available plasmid pcDNAI, pcDNA3, or PEF6/v5-His(Invitrogen, San Diego, Calif.) may, for example, be used for productionin mammalian cells such as Cos-7 and CHO cells. One having ordinaryskill in the art can use these commercial expression vectors and systemsor others to produce proteins by routine techniques and readilyavailable starting materials. (See e.g., Sambrook et al., eds., 2001,supra) Thus, the desired proteins or fragments can be prepared in bothprokaryotic and eukaryotic systems, resulting in a spectrum of processedforms of the protein or fragments.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts (See e.g.,Sambrook et al., eds., 2001, supra).

In some embodiments, the nucleic acid molecules can also be prepared asa genetic construct. “Genetic constructs” include regulatory elementsnecessary for gene expression of a nucleic acid molecule. The elementsinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers can be used for geneexpression of the sequence that encodes the protein or fragment. It isnecessary that these elements be operably linked to the sequence thatencodes the desired polypeptide and that the regulatory elements areoperably in the individual or cell to whom they are administered.Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual or cellto which the gene construct is administered. The initiation andtermination codons must be in frame with the coding sequence. Promotersand polyadenylation signals used must be functional within the cells.Examples of promoters useful to practice the present invention includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metallothionein. Examples of polyadenylationsignals useful to practice the present invention include but are notlimited to SV40 polyadenylation signals and LTR polyadenylation signals.In some embodiments, the SV40 polyadenylation signal which is inpCEP4plasmid (Invitrogen, San Diego Calif.), referred to as the SV40polyadenylation signal, is used. In addition to the regulatory elementsrequired for DNA expression, other elements may also be included in theDNA molecule. Such additional elements include enhancers. The enhancermay be selected from the group including but not limited to: humanActin, human Myosin, human Hemoglobin, human muscle creatine and viralenhancers such as those from CMV, RSV and EBV. Genetic constructs can beprovided with mammalian origin of replication in order to maintain theconstruct extrachromosomally and produce multiple copies of theconstruct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (SanDiego, Calif.) contain the Epstein Barr virus origin of replication andnuclear antigen EBNA-1 coding region which produces high copy episomalreplication without integration. In some embodiments, the nucleic acidmolecule is free of infectious particles.

In some embodiments, the present invention provides compositionscomprising at least one polypeptide fragment of ADAMTS-1 that inhibitscell proliferation or cell growth or metastasis. In some embodiments,the composition comprises a fragment comprising SEQ ID NO:1 and/or SEQID NO:2. In some embodiments, the composition comprises a fragmentcomprising SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, and/or SEQ ID NO: 11.In some embodiments, the composition comprises two or at least twopolypeptide fragments of ADAMTS-1. In some embodiments, the fragmentcomprises the TSP-type I motif. In some embodiments, the composition isa pharmaceutical composition.

As used herein, the term “fragment of ADAMTS-1 that inhibits cellproliferation or metastasis” refers to a fragment of ADAMTS-1 that caninhibit cell growth, cell division, or cell proliferation. In someembodiments, the fragment inhibits cell growth, cell division, cellproliferation, or metastasis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 100%. In some embodiments, the fragment can inhibit cellgrowth, cell division, cell proliferation, or metastasis by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%. One of skill in the art can determine the level of inhibition by,for example, comparing the property or properties of the cell orpopulation of cells in the absence of the fragment.

A fragment of ADAMTS-1 that inhibits cell proliferation, cell growth,cell division, or metastasis can be identified using any known growth,proliferation, or division assay. For example, one of skill in the artcan contact a fragment of ADAMTS-1 with a cell either in vitro or invivo and determine whether the cell's growth, proliferation, divisions,or metastasis have been inhibited. One of skill in the art could alsouse a nucleic acid molecule encoding a fragment of ADAMTS-1 andintroduce the nucleic acid molecule into a cell or organism such thatthe fragment is expressed. The cell's or population of cell's propertyor properties could then be measured and it could be determined whetherthe fragment encoded by the nucleic acid molecule can inhibit cellgrowth (proliferation), cell division, or metastasis.

The fragments of ADAMTS-1 that can be used to inhibit cell proliferationor growth can be produced by a cleavage event of ADAMTS-1. In someembodiments, the cleavage produces fragments of ADAMTS-1 comprising SEQID NOs: 5, 7, 9 and/or 11 or encoded by nucleic acid moleculescomprising SEQ ID NO: 6, 8, 10, and/or 12.

In some embodiments, the present invention provides methods ofinhibiting the cleavage of ADAMTS-1 in a cell comprising contacting thecell with a cleavage inhibiting factor. A “cleavage inhibiting factor”is a compound or composition that can inhibit the cleavage of ADAMTS-1.In some embodiments, the cleavage of ADAMTS-1 is auto-cleavage orcleavage that is facilitated by a protease that is not ADAMTS-1. In someembodiments, the cleavage inhibiting factor is heparin or heparansulfate proteoglycans (HSPGs). Heparin or derivatives of heparin werefound to inhibit the cleavage of ADAMTS-1 as described herein. In someembodiments, the present invention provides methods of promotingcleavage of ADAMTS-1 comprising contacting ADAMTS-1 with a ADAMTS-1cleavage activating factor. In some embodiments, the cleavage activatingfactor is a compound that inhibits and/or sequesters heparin. In someembodiments, the factor that inhibits heparin is heparinase, plateletfactor 4 (PF4-a), protamine, or polybrene. A “cleavage activatingfactor” is a compound or composition that enhances, induces, orincreases the level of cleavage of ADAMTS-1. In some embodiments, thecleavage of ADAMTS-1 can be auto-cleavage. In some embodiments, thecleavage of ADAMTS-1 can be facilitated by a protease that is notADAMTS-1.

In some embodiments, the present invention provides methods ofinhibiting cell proliferation or metastasis comprising contacting thecell with a fragment of ADAMTS-1 that inhibits cell proliferation ormetastasis. In some embodiments, the fragment of ADAMTS-1 comprises afragment of SEQ ID NO:1 and/or SEQ ID NO:3. In some embodiments, thefragment comprises SEQ ID NOs: 5, 7, 9 and/or 11. One of skill in theart can determine if the fragment inhibits cell proliferation ormetastasis of a cell or population of cells by measuring the growth ormetastasis in the presence and/or absence of the fragment of ADAMTS-1.

As used herein, the term “cell” refers to any cell. In some embodiments,the cell is a human cell or a mouse cell. In some embodiments, the cellis a tumor cell, inflammatory cells, or keratinocytes. In someembodiments, the cell is a primary tumor cell. As used herein, the term“primary tumor cell” refers to a cell that has been excised from a tumorfrom an individual or animal and has not been propagated through morethan 10 cell divisions.

The discovery that fragments of ADAMTS-1 can inhibit cell growth and/ormetastasis demonstrates that in some embodiments, the present inventionprovides methods of treating cancer in an individual comprisingadministering to the individual a therapeutically effective amount of afragment of ADAMTS-1 that is able to inhibit cell proliferation ormetastasis. The fragments can also be said to inhibit tumor growth andthe like. In some embodiments, the fragment comprises a fragment of SEQID NO:1 and/or SEQ ID NO: 3. In some embodiments, the fragment comprisesSEQ ID NOs: 5, 7, 9 and/or 11. In some embodiments, the fragment ofADAMTS-1 is co-administered with at least one other cancer treatment.The fragment of ADAMTS-1 can be either administered prior to,subsequently to, or at the same time as the other cancer treatment. Thefragment(s) of ADAMTS-1 can be co-administered with any other cancertreatment, including, but not limited to, surgery, chemotherapy,antibodies, small molecules, radiation, and the like. In someembodiments, the fragment of ADAMTS-1 that is used to treat the cancerin an individual is a fragment of ADAMTS-1 that is able to inhibit cellproliferation, metastasis, or angiogenesis. In some embodiments, thefragment inhibits cell proliferation and/or metastasis in vitro.

Since it has been discovered that the full length ADAMTS-1 is pro-cancerwhile the cleavage fragments of ADAMTS-1 have anti-cancer properties,the present invention provides methods of treating cancer in anindividual comprising administering to the individual a composition thatinduces the cleavage of ADAMTS-1. In some embodiments, the compositionthat induces the cleavage of ADAMTS-1 is a heparin inhibitor. Examplesof heparin inhibitors include, but are not limited to, heparinase,platelet factor 4 (PF4-a), protamine, polybrene, the heparin-bindingdomain/peptide derived from HSPGs, and the like. In some embodiments,the cleavage of ADAMTS-1 results in the production of at least onefragment of ADAMTS-1 that can inhibit cell proliferation or metastasis.In some embodiments, the cleavage of ADAMTS-1 results in the productionof two or at least two fragments of ADAMTS-1 that can inhibit cellproliferation or metastasis. In some embodiments, the fragments that areproduced by the cleavage of ADAMTS-1 comprise SEQ ID NOs: 5, 7, 9 and/or11.

In some embodiments, the present invention provides methods ofinhibiting metastasis in an individual comprising administering theindividual a fragment or mutant of ADAMTS-1 that inhibits metastasisand/or angiogenesis. In some embodiments, the mutant of ADAMTS-1 is ametalloproteinase defective mutant. In some embodiments, the fragment ofADAMTS-1 that inhibits metastasis comprises SEQ ID NO: 5, 7, 9, and/or11. In some embodiments, the fragment or mutant of ADAMTS-1 thatinhibits metastasis, cell growth or proliferation and/or angiogenesiscomprises SEQ ID NO: 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33,and/or 35.

In some embodiments, a method of treating cancer can refer to a methodof inhibiting cell growth, division, inducing cell death (e.g. apoptosisand/or necrosis), promoting metastasis and angiogenesis, or combinationsthereof.

The fragments or mutants of the present invention can also beadministered in the form of a nucleic acid molecule that encodes for thefragments or for the mutants. In some embodiments, the nucleic acidmolecule comprises SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, and/or 36.

The present invention also provides antibodies or fragments ofantibodies that can specifically bind to and block the pro-canceractivity of ADAMTS-1. In some embodiments, the antibody specificallybinds to ADAMTS-1 comprising SEQ ID NO: 1, 2, 3, or 4

As used herein, the term “specifically binds to” in reference to anantibody refers to an antibody that will bind to one peptide or proteinwith higher affinity than another peptide. In some embodiments, theantibody that specifically binds to a peptide or polypeptide will notbind to more than one peptide. In some embodiments, the specificantibody binds with a K_(d) that is 10×, 100×, 1000× greater to onepeptide over another. Methods of making and identifying specificantibodies are routine.

The present invention also provides for antibodies that binds tofull-length ADAMTS-1 to inhibit cell proliferation, division, growth, ormetastasis. In some embodiments, the polypeptide comprises SEQ ID NO: 1,2, 3, or 4.

The present invention also provides methods of inducing the cleavage ofADAMTS-1 in a cell comprising contacting the cell with a heparininhibitor. Examples of heparin inhibitors include, but are not limitedto heparinase, platelet factor 4 (PF4-a), protamine, polybrene, and thelike.

The present invention also provides for methods for identifying aninhibitor or an activator of ADAMTS-1 cleavage comprising performing atest assay comprising contacting ADAMTS-1 with a test compound; andmeasuring the cleavage of ADAMTS-1, wherein a decrease in cleavageindicates that the test compound is a cleavage inhibitor or wherein anincrease in cleavage indicates that the test compound is a cleavageactivator. In some embodiments, the effect of the test compound iscompared what occurs in the absence of any test compound. In someembodiments, the compound is contacted with ADAMTS-1 under conditions inwhich ADAMTS-1 is cleaved. In some embodiments, ADAMTS-1 undergoesauto-cleavage (e.g. where the enzyme cleaves itself). In someembodiments, the method comprising contacting a test compound withADAMTS-1 under conditions where ADAMTS-1 can be cleaved. Theseconditions can be any conditions and can be modified such that ADAMTS-1is able to be cleaved either by itself (auto-cleavage) or by anothermolecule. Conditions that can be modified include, but are not limitedto, pH, ion concentration, metal concentration, and the like.

In some embodiments the methods comprise contacting more than one testcompound, in parallel. In some embodiments, the methods comprisescontacting 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100,1000, at least 2, at least 5, at least 10, at least 50, at least 100, orat least 1000 test compounds in parallel. In some embodiments, thepresent invention is used in High Throughput Screening of compounds andcomplete combinatorial libraries can be assayed, e.g., up to thousandsof compounds. Methods of how to perform high throughput screenings arewell known in the art. The methods can also be automated such that arobot can perform the experiments. The present invention can be adaptedfor the screening of large numbers of compounds, such as combinatoriallibraries of compounds. Indeed, compositions and methods allowingefficient and simple screening of several compounds in short periods oftime are provided. The instant methods can be partially or completelyautomated, thereby allowing efficient and simultaneous screening oflarge sets of compounds.

In some embodiments, the method of the present invention comprises thestep of contacting a cell expressing v5-epitope tagged ADAMTS-1 (such asTA3_(ADAMTS-1)) in the presence of a test compound. The cells can beobserved to determine if the test compound inhibits or promotes thecleavage of ADAMTS-1. A control may be provided in which the cell is notcontacted with a test compound. A further control may be provided inwhich test compound is contacted with cells that either do not expressADAMTS-1 or in which ADAMTS-1 cannot be cleaved (the cleavage-resistantADAMTS-1 mutant). If the cells contacted with the test compound increasethe cleavage of ADAMTS-1 then pro-cleavage activity is indicated for thetest compound. If the cells contacted with the test compound decreasethe cleavage of ADAMTS-1 then anti-cleavage activity is indicated forthe test compound.

Positive and negative controls may be performed in which known amountsof test compound and no compound, respectively, are added to the assay.One skilled in the art would have the necessary knowledge to perform theappropriate controls.

The test compound can be any product in isolated form or in mixture withany other material (e.g., any other product(s)). The compound may bedefined in terms of structure and/or composition, or it may beundefined. For instance, the compound may be an isolated andstructurally-defined product, an isolated product of unknown structure,a mixture of several known and characterized products or an undefinedcomposition comprising one or several products. Examples of suchundefined compositions include for instance tissue samples, biologicalfluids, cell supernatants, vegetal preparations, etc. The test compoundmay be any organic or inorganic product, including a polypeptide (or aprotein or peptide), a nucleic acid, a lipid, a polysaccharide, achemical product, or any mixture or derivatives thereof. The compoundsmay be of natural origin or synthetic origin, including libraries ofcompounds.

In some embodiments, the activity of the test compound(s) is unknown,and the method of this invention is used to identify compoundsexhibiting the selected property (e.g., ADAMTS-1 cleavage). However, insome embodiments instances where the activity (or type of activity) ofthe test compound(s) is known or expected, the method can be used tofurther characterize the activity (in terms of specificity, efficacy,etc.) and/or to optimize the activity, by assaying derivatives of thetest compounds.

The amount (or concentration) of test compound can be adjusted by theuser, depending on the type of compound (its toxicity, cell penetrationcapacity, etc.), the number of cells, the length of incubation period,the amount of ADAMTS-1, etc. In some embodiments, the compound can becontacted in the presence of an agent that facilitates penetration orcontact with a cell comprising ADAMTS-1. The test compound is provided,in some embodiments, in solution. Serial dilutions of test compounds maybe used in a series of assays. In some embodiments, test compound(s) maybe added at concentrations from 0.01 μM to 1M. In some embodiments, arange of final concentrations of a test compound is from 10 μM to 100μM. One such test compound that is effective to activate cleavage ofADAMTS-1 in a cell is a heparin inhibitor.

In some embodiments, the method comprises measuring ADAMTS-1 cleavage inthe presence of the test compound. If the test compound is found tocleave ADAMTS-1 it is indicative that the test compound is pro-cleavageADAMTS-1 agent. Since the cleavage fragments of ADAMTS-1 agent, apro-cleavage fragment can also be considered an anti-cancer agent.

ADAMTS-1 cleavage can be measured by any means that demonstrates thatthe cleavage of ADAMTS-1 has been modulated (increased or decreased) inthe presence of the test compound. Examples of how to measure ADAMTS-1cleavage include measuring an increase or decrease in the cleavagefragments of ADAMTS-1. The cleavage fragments can be measured by anymeans including, but not limited to, Western Blot, ELISA, SandwichAssay, and the like. Methods of measuring the levels protein cleavagefragments are routine to one of ordinary skill in the art.

In some embodiments, the test compound activates the cleavage ofADAMTS-1 by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 200%. In some embodiments, the test compoundinhibits the cleavage of ADAMTS-1 by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 99%. In some embodiments, thecleavage of ADAMTS-1 is compared to cleavage of ADAMTS-1 observed in theabsence of the test compound.

In some embodiments, the methods further comprise performing a controlassay. In some embodiments, the control assay comprising contacting acell with a negative or positive control and measuring, including, butnot limited to, ADAMTS-1 cleavage. In some embodiments, the controlcompound is compared to the test compound. In some embodiments, thecontrol compound is a negative control (e.g. a compound that does notinhibit or activate ADAMTS-1 cleavage). A negative control can also bethe absence of a test compound or the vehicle (e.g. solvent) that thetest compound is contacted with the cell. In some embodiments, thecontrol compound is a positive control (e.g. a compound that inhibits oractivates ADAMTS-1 cleavage).

In some embodiments, the test compound is a small molecule, a peptide(including the peptides from the heparin-binding proteins and HSPGs), anantibody, a cellular fraction, a protease, or a mixture thereof. Asdiscussed above, the test compound can be contacted with a cellcomprising ADAMTS-1, but the test compound can be contacted withADAMTS-1. For example, ADAMTS-1 can be expressed as a protein and eitherbe purified or not be purified, but is isolated from a cell. For thepurposes of the screening assays to identify test compounds that caninhibit or activate the cleavage of ADAMTS-1, an isolated protein is aprotein that is separated from a cell. The protein can be purified fromother components in the cell, but it does not have to be. In someembodiments, an isolated ADAMTS-1 protein results from a cell beinglysed which releases all the contents of the cell. The cleavage ofADAMTS-1 can then be measured or monitored in a non-cellularenvironment. The test compound is then contacted with ADAMTS-1 todetermine if the test compound can inhibit or activate the cleavage ofADAMTS-1.

In some embodiments, the methods further comprise performing a negativecontrol assay which comprises contacting a cell that does not compriseADAMNTS-1 or a cell that comprises a cleavage resistant mutant ofADAMTS-1. In some embodiments, the negative control assay comprisescontacting an isolated cleavage resistant mutant of ADAMTS-1.

The present invention also provides methods for identifying ananti-cancer agent comprising performing a test assay comprisingcontacting a cell comprising ADAMTS-1 with a test compound; andmeasuring the cleavage of ADAMTS-1, wherein an increase in cleavageindicates that the test compound is an anti-cancer compound. In someembodiments, the cleavage in the presence of the test compound iscompared to an assay where the cell is comprising ADAMTS-1 is notcontacted with the test compound.

As used herein, “a cell comprising ADAMTS-1” refers to a cell expressingthe protein ADAMTS-1. The cell can be either be expressing the proteinendogenously (e.g. from within its native genome) or exogenously. Anexogenously expressed protein is a protein in a cell that would notnormally be present except for some modification. The exogenouslyexpressed protein can be, for example, transfected into a cell eitherstably or transiently.

The present invention also provides methods of inhibiting angiogenesisin an individual comprising administering to the individual a fragmentof ADAMTS-1. In some embodiments, the fragment of ADAMTS-1 comprisesADAMTS-1_(CTCF) or ADAMTS-1_(NTCF) (SEQ ID NOs: 5, 7, 9 and/or 11). Insome embodiments a nucleic acid molecule encoding the fragments isadministered. In some embodiments, the nucleic acid molecule comprisesSEQ ID NOs: 6, 8, 10, and/or 12.

The present invention provides methods of inhibiting the growth ormetastasis of a tumor. In some embodiments, the tumor is vascularized ornon-vascularized.

The present invention also provides methods of treating cancercomprising inhibiting the metalloproteinase activity of ADAMTS-1. Insome embodiments, the metalloproteinase activity of ADAMTS-1 isinhibited by administering a metalloproteinase defective full-lengthADAMTS-1 or the ADAMTS-1 fragments containing its substrate-bindingdomain such as ADAMTS-1_(spacer/Cys-rich) or ADAMTS-1_(spacer), whichcan act as the dominant negative mutants of ADAMTS-1 and inhibit theactivity of the wild-type protein. In some embodiments, themetalloproteinase defective ADAMTS-1 comprises SEQ ID NO: 29, 31, 33,and/or 35. In some embodiments, the metalloproteinase activity isinhibited by an antibody or a small molecule that binds to ADAMTS-1. Insome embodiments, the metalloproteinase activity is inhibited by anantibody or a small molecule that binds to the metalloproteinase activesite of ADAMTS-1.

The present invention also provides methods of identifying inhibitors ofADAMTS-1 metalloproteinase activity comprising contacting a fragment ofor full-length ADAMTS-1 that has metalloproteinase activity with a testcompound and determining if the metalloproteinase activity is inhibited.(In some embodiments, the fragment of ADAMTS-1 comprises SEQ ID NO: 5,7, 9, and/or 11.) In some embodiments, the activity in the presence ofthe test compound is compared to the activity in the absence of the testcompound. In some embodiments, the method comprises comparing theactivity with a positive control assay and/or a negative control assay.In some embodiments, the method comprises comparing the activity of thefragment to a fragment that is defective in metalloproteinase activity.A fragment can be defective in metalloproteinase because of a mutation,substitution, deletion, or insertion. In some embodiments, the fragmentis defective in metalloproteinase activity due to a Glu to Gln mutation.In some embodiments, the fragment that lacks metalloproteinase activitycomprises SEQ ID NO: 33 and/or 35. In some embodiments, the fragmentthat lacks metalloproteinase activity is encoded by a nucleic acidmolecule comprising SEQ ID NO: 34 and/or 36.

Methods of measuring metalloproteinase activity (e.g. ADAMTS-1 activity)are routine. For example, the cleavage of substrates of ADAMTS-1 can bemeasured and compared in the absence and presence of a test compound.However, any method or means can be used to measure metalloproteinaseactivity of ADAMTS-1. Substrates of ADAMTS-1 are known in the art andcan be measured. In some embodiments, the substrate of themetalloproteinase is aggrecan or versican.

In some embodiments, the present invention provides methods of treatingcancer comprising administering to an individual a compound that is aADAMTS-1 metalloproteinase activity inhibitor. In some embodiments, theinhibitor is a dominant negative mutant of ADAMTS-1. In someembodiments, the inhibitor is a polypeptide or comprising SEQ ID NO: 33and/or 35. In some embodiments, the inhibitor is encoded by a nucleicacid molecule comprising SEQ ID NO: 34 and/or 36.

Other fragments or mutants of ADAMTS-1 can also be used to treat cancerbecause they also act as a dominant negative regulator of ADAMTS-1 and,thus, be able to inhibit the function of ADAMTS-1. Accordingly, thepresent invention provides methods of treating cancer comprisingadministering a therapeutically effective amount of a compositioncomprising a fragment of ADAMTS-1 comprising the spacer/Cys-rich and/orSpacer domain of ADAMTS-1.

The present invention also provides polypeptide fragments of ADAMTS-1comprising the spacer/Cys-rich and/or spacer domain of ADAMTS-1. In someembodiments, the fragment comprises SEQ ID NO: 17, 19, 21, and/or 23. Insome embodiments, the fragments are encoded by nucleic acid moleculescomprising 18, 20, 22, and/or 24.

The present invention also provides for fragments of ADAMTS-1 that bindto the extracellular matrix (ECM). According, in some embodiments, thepresent invention provides an ECM binding fragment of ADAMTS-1. An “ECMbinding fragment of ADAMTS-1” is a fragment of ADAMTS-1 that binds tothe ECM. In some embodiments, the ECM binding fragment of ADAMTS-1comprises SEQ ID NO: 17, 19, 21, and/or 23. In some embodiments, anucleic acid molecule encodes for an ECM binding fragment of ADAMTS-1.In some embodiments, the ECM binding fragment comprises SEQ ID NO: 18,20, 22, and/or 24.

In some embodiments, the present invention provides nucleic acidmolecules encoding any fragment of ADAMTS-1 described herein. In someembodiments, the nucleic acid molecule comprises SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 16, 28, 30, 32, 34, 36, or combinationsthereof.

In some embodiments, the present inventions provides polypeptidescomprising at least a fragment of ADAMTS-1 as described herein. In someembodiments, the polypeptides comprise SEQ ID NO: 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 33, 35, or combinations thereof. In someembodiments, the present invention provides polypeptides that comprisemutations that inactivate the metalloproteinase activity of ADAMTS-1. Insome embodiments, the mutation is a Glu to Gln mutation that correspondsto position 386 (mouse) (or 385 in human) of the wild-type ADAMTS-1. Insome embodiments, the mutant ADAMTS-1 comprises SEQ ID NO: 29 and/or 31.In some embodiments a nucleic acid molecule encoding such mutants isprovided. In some embodiments, the nucleic acid molecule comprises SEQID NO: 30 and/or 32.

The present invention also provides methods for identifying a compoundthat induces the cleavage of ADAMTS-1 comprising performing a test assaycomprising identifying a compound that inhibits heparin, wherein if acompound inhibits heparin, the compound would be said to induce thecleavage of ADAMTS-1. Since heparin inhibitors induce the cleavage ofADAMTS-1 heparin inhibitors would be able to activate the cleavage ofADAMTS-1. Thus, a compound that is identified as heparin inhibitor wouldbe said to be able to induce the cleavage of ADAMTS-1. In someembodiments, the effect of heparin is a protective effect.

The present invention also provides methods of identifying a heparininhibitor comprising contacting heparin and ADAMTS-1 with a testcompound under conditions that ADAMTS-1 is cleaved in the absence ofheparin and determining if the test compound inhibits heparin. Asdescribed herein, heparin inhibits the cleavage of ADAMTS-1. Therefore,a test compound that inhibits heparin will allow ADAMTS-1 to be cleavedby another protein or by itself. A test compound is said to be a heparininhibitor if ADAMTS-1 is cleaved in the presence of heparin. In someembodiments, the heparin and ADAMTS-1 are free of cellular proteins. Insome embodiments, the heparin and ADAMTS-1 is free of extracellularmatrix.

A fragment of ADAMTS-1, a nucleic acid molecule encoding a fragment ofADAMTS-1, a compound that inhibits or activates the cleavage of ADAMTS-1can be administered by any means to the individual whether in the formof a composition or a pharmaceutical composition. Methods ofadministration are known to one of skill in the art. For example, theagent can be prepared as a pharmaceutical composition. In someembodiments, the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier or diluent. In some embodiments, the pharmaceuticalcompositions are sterile and/or pyrogen free. The pharmaceuticalcomposition comprising the molecule and a pharmaceutically acceptablecarrier or diluent may be formulated by one having ordinary skill in theart with compositions selected depending upon the chosen mode ofadministration. Suitable pharmaceutical carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, A. Osol, astandard reference text in this field.

For parenteral administration, the composition can be, for example,formulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Examples of such vehicles are water, saline, Ringer's solution, dextrosesolution, and 5% human serum albumin. Liposomes and nonaqueous vehiclessuch as fixed oils may also be used. The vehicle or lyophilized powdermay contain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as a single doses or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions may be administered by any means thatenables the agent to reach the agent's site of action in the body of amammal. Because the compositions may be subject to being digested whenadministered orally, parenteral administration, i.e., intravenous,subcutaneous, intramuscular, would ordinarily be used to optimizeabsorption. In addition, the pharmaceutical compositions of the presentinvention may be injected at a site at or near hyperproliferativegrowth. For example, administration may be by direct injection into asolid tumor mass or in the tissue directly adjacent thereto. Thecomposition may also be formulated with a pharmaceutically acceptabletopical carrier and the formulation may be administered topically as acreme, lotion or ointment for example.

The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment. Usually, adaily dosage of a composition to treat cancer is used in an amounteffect to have an anti-cancer effect. In some embodiments, the dailydosage is used in an amount to cleave ADAMTS-1 into a fragment that caninhibit cell proliferation or cell growth (e.g tumor growth). In someembodiments, the dosage can be about 1 lag to 100 milligrams perkilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein. Those of skill in the art will readily recognize a variety ofnon-critical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Materials and Methods

Cell Lines and Reagents

Human umbilical vein endothelial cells (HUVECs) were obtained fromCambrex (Walkersville, Md.). TA3 transfectants were maintained asdescribed previously (11, 12). Anti-v5 epitope (Invitrogen), -vWF(Dako), -phosphorylated tyrosine (BD Transduction Lab), -EGFR, -ErbB-2,-Erk1/2, and -phospho-Erk1/2 (Santa Cruz) antibodies, and Brdu-cellproliferation kit (Roche) and Apoptag kit (Chemicon) were used in theexperiments.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Mutagenesis,and Expression Constructions

Expression of ADAMTS-1 was assessed by RT-PCR as described (13).Full-length mouse ADAMTS-1 was obtained by RT-PCR with a primer pairconsisting of 24 nucleotides corresponding to the 3′ or 5′ extremity ofthe coding sequence of ADAMTS-1 (accession number NM_(—)009621). Thestop codon was omitted from the reverse primers to fuse ADAMTS-1 to theC-terminal v5 epitope tag existed in the expression vector(pEF6/v5-HisTOPO, Invitrogen). Various mutation and deletion of ADAMTS-1were generated as detailed in FIG. 1A using the QuikChange™ site andExSite PCR-based site-directed mutagenesis kits (Stratagene).

Transfection

Lipofectamine (Invitrogen) was used to transfect TA3_(wt1) cells withempty expression vector alone or the expression constructs containingcDNA inserts encoding ADAMTS-1 and various mutant or fragments ofADAMTS-1 (FIG. 1A). TA3 transfectants were selected and the expressionlevel of v5-tagged full-length and fragments of ADAMTS-1 was determinedby Western blotting with anti-v5 antibody (Invitrogen).

ADAMTS-1 Production and Purification, Proteolytic Cleavage Assay, andWestern Blot Analysis

Cell culture supernatants derived from Cos-7 and TA3 transfectantsexpressing v5-epitope tagged wild type ADAMTS-1 or ADAMTS-1 mutants(FIG. 1A) were collected and purified through Ni⁺-Probond (Invitrogen)and anti-v5 antibody conjugated affinity columns (Sigma).Auto-proteolytic cleavage capacity of ADAMTS-1 was assessed by in vitroproteolytic cleavage assay using purified ADAMTS-1. In this assay, 100ng of ADAMTS-1 was incubated in 50 mM Tris-acetate buffer (pH 7.3)containing 5 mM CaCl₂ and 0.1M NaCl at 37° C. for 30 min, 1, 2, 4, 8 and12 hours, and reaction was stopped by addition of 8×SDS sample buffer.The reaction products were analyzed by Western blot with anti-v5 mAb.

To assess ADAMTS-1 cleavage in cellular context and to determine how thecleavage is regulated, Cos-7 or TA3 transfectants expressing ADAMTS-1 orADAMTS-1E/Q was cultured for 48 hours in the absence or presence ofdifferent reagents as detailed in the figure legend, and the cellculture supernatants were collected and analyzed by Western blot withanti-v5 antibody.

Tumor Cell Tracking and Pulmonary Metastasis

To track TA3 transfectants during early pulmonary metastasis, the TA3transfectants were labeled with green 5-chloromethyl-fluoresceindiacetate (CMFDA, Molecular Probes, Inc.) as described (13), and theCMFDA-labeled TA3 transfectants (1×10⁶ cells/mouse) were injected intothe tail vein of A/Jax syngenic mice (the Jackson Lab). The mice weresacrificed 24 hours after the injection, and lungs were removed, fixed,and sectioned. The localization of tumor cells in mouse lung parenchymawere revealed under fluorescence microscope, and the extent of tumorcell extravasation was determine by counting number of the tumor cellsin five randomly selected 10× microscopic fields.

Experimental pulmonary metastasis was carried out as detailed previously(13), and five independent clonal TA3 transfectants expressing ADAMTS-1,ADAMTS-1_(CTCF), ADAMTS-1_(NTCF) or ADAMTS-1_(minusTSP), or transfectedwith the empty expression vector were used. For each type of theexperiment, six mice were injected with each clonal transfectant and twoindependent experiments were performed. The experimental mice wereobserved daily after the i.v. injection and duration of mouse survivalwas recorded. The survival rate of these mice was calculated as thefollowing: survival rate (%)=(number of mice are still alive/totalnumber of the experimental mice)×100%. The mice that are free of symptom60 days after the i.v. injection were sacrificed and their lungs wereexamined. In the second set of experiments, 11 and 18 days after i.v.injection, pulmonary metastatic burden was assessed by measuring weightof the mouse lungs.

Histology and Immunohistochemistry

To determine the tumor cell proliferation rate in vivo,5-Bromo-2′-deoxy-uridine (Brdu) was injected into mice four hours priorto sacrifice of the experimental mice. The mouse lungs were fixed,sectioned, and stained with H&E as described (11). In addition, thesections were reacted with anti-von Willebrand factor (vWF) antibody toassess tumor angiogenesis, with anti-Brdu antibody to detectproliferating cells or with Apoptag kit to detect apoptotic cells insitu. Total number of the tumor cells and number of the tumor cells thatare positive for anti-Brdu antibody or TUNEL-staining were counted infive randomly selected 400× microscopic fields within the pulmonarymacro- and micro-metastases. More than 2,000 cells were counted in totalfor each type of transfectants. The proliferation and apoptosis rate wascalculated as the following: proliferation or apoptosis rate=(number ofthe anti-Brdu or TUNEL-positive cells per microscopic field/total numberof the tumor cells per microscopic field)×100%. To determine bloodvessel number, the vWF-positive blood vessels were countered in sixrandomly selected 200× microscopic fields within macro- ormicro-metastases. The number of blood vessels/microscopic field wasexpressed as means+/−S.D.

EGFR and ErbB-2 Phosphorylation

RIPA buffer (50 mM Tris-HCl, PH 7.4, 50 mM NaCl, 1% Triton-X100, 2 mMEDTA, 2 mM sodium orthovanadate, 2 mM sodium fluoride, 2 mMphenylmethylsulfonyl fluoride, 1 mM Leupeptin, 1 mM Pepstain A, and 10μg/ml aprotinin) was used to extract the lung tissues derived from themice that were injected with or without different TA3 transfectants(1×10⁶/mouse) intravenously 24 hours prior. The proteins were used inthe immunoprecipitation reactions to pull-down EGFR and ErbB-2 using theagarose beads conjugated with anti-EGFR or anti-ErbB-2 antibody (SantaCruz). The precipitated proteins were analyzed by Western blotting withanti-phosphotyrosine antibody (BD Bioscience) to detect phosphor-EGFRand phosphor-ErbB-2, or with anti-EGFR or anti-ErbB-2 antibody (SantaCruz) to detect total amount of EGFR or ErbB-2, respectively.

Shedding of the EGF Family GFs and Activation of Erk1/2 Kinases

Shedding of the transmembrane precursors of AR, HB-EGF, and epigen byADAMTS-1, its mutant and fragments were assessed by co-transfection ofCos-7 cells with the expression constructs containing cDNA inserts thatencode these EGF family precursors and various ADAMTS-1 constructs asdetailed in the figure legend. The concentrated cell culturesupernatants of the co-transfected COS-7 cells were analyzed by Westernblotting to detect the soluble GFs using the corresponding antibodies(R&D Systems).

Ability of the ADAMTS-1 fragments to inhibit activation of Erk1/2 kinaseinduced by soluble AR (5 ng/ml) and HB-EGF (4 ng/ml) were assessed byapplying the serum starved MCF-10A cells with purified soluble AR orHB-EGF in the absence or presence of their corresponding neutralizationantibodies or purified full-length ADAMTS-1 or the ADAMTS-1 fragments(400 ng). MCF-10A cells were then lysed and equal amount of the proteinswere analyzed by Western blotting with anti-phospho-Erk1/2 to detectphosphor-Erk1/2 or with anti-Erk antibody to detect total amount ofErk1/2 protein.

HUVECs were cultured until subconfluence and switched to serum-freemedium (SFM) for overnight. VEGF₁₆₅ (10 ng), bFGF (10 ng), AR (5 ng),and HB-EGF (4 ng) were applied to the serum-starved HUVECs in theabsence or presence of 400 ng of ADAMTS-1, ADAMTS-1_(minusTSP),ADAMTS-1_(NTCF), or ADAMTS-1_(CTCF) for 20 minutes. The HUVECs werelysed and equal amount of the proteins were subjected to Westernblotting with anti-phospho-Erk1/2 or anti-Erk (Santa Cruz) to detectphosphor-Erk1/2 or total amount of Erk, respectively.

Example 2 ADAMTS-1 Undergoes Auto-Proteolytic Cleavage and theSelf-Cleavage of ADAMTS-1 is Regulated

Previous results have shown that ADAMTS-1 is cleaved within the spacerregion and several matrix metalloproteinases (MMPs) are responsible forthe cleavage (9). Since several other members of ADAMTS family undergoauto-proteolytic cleavage and ADAMTS-1 is an active metalloproteinase,the possibility that the cleavage of ADAMTS-1 can be mediated by its ownmetalloproteinase activity was investigated. To achieve that, aprotease-dead mutant of ADAMTS-1 was generated by switching E₃₈₆ to Q(ADAMTS-1E/Q) in the Zinc-binding pocket of the metalloproteinasedomain. The expression constructs containing v5-epitope tagged wild typeADAMTS-1 or ADAMTS-1E/Q were used to transfect Cos-7 cells. The cellculture supernatants of the transiently transfected Cos-7 cells wereanalyzed and the results showed that only wild type ADAMTS-1 but notADAMTS-1E/Q is cleaved to generate the C-terminal cleavage fragments(FIG. 1B, arrows), suggesting that the metalloproteinase activity ofADAMTS-1 is required for the cleavage.

In order to produce full-length ADMATS-1, the regulation of ADAMTS-1cleavage was investigated. Different reagents were applied to a stableTA3 transfectant expressing ADAMTS-1, and the cell culture supernatantswere analyzed 48 hours late. The result showed that heparin and heparansulfate (HS) completely block the proteolytic cleavage of ADAMTS-1 (FIG.1C), while the control glycosaminoglycans (GAGs), chondroitin sulfate(CS) and hyaluronan (HA), and displayed no effect on the cleavage. Thisresult suggests that auto-proteolytic cleavage of ADAMTS-1 is regulatedby synthesis and degradation rate of HS/heparan sulfate proteoglycans(HSPGs) in the microenvironment where ADAMTS-1 is produced and HS/HSPGslikely play important role in regulating ADAMTS-1 function.

Full-length ADAMTS-1 protein was produced by Cos-7 cells transfectedwith the expression construct containing ADAMTS-1 cDNA in the presenceof heparin. Cell culture media of the transfected Cos-7 cells werecollected and purified through the affinity columns. Purified ADAMTS-1was used in a proteolytic cleavage assay and the result showed thatADAMTS-1 was auto-proteolytically cleaved to release v5-epitope taggedC-terminal cleavage fragments that have molecular weight similar to thatgenerated in the cell culture condition (FIG. 1E).

ADAMTS-1 Promotes Metastasis, while ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)Block the Process

ADAMTS-1 was found to inhibit bFGF-induced vascularization in the corneapocket assay and VEGF-induced angiogenesis in the chorioallantoicmembrane assay and tumor growth in vivo. However, a study analyzingclinical pancreatic cancer samples demonstrated that increasedexpression of ADAMTS-1 is correlated to enhanced metastatic potentialand worse prognosis, implying that ADAMTS-1 facilitates tumormetastasis. In addition, studies have shown that ADAMTS-1 is one of thegenes up-regulated in the breast cancer with elevated metastaticactivity. To determine the exact roles of ADAMTS-1 and its cleavagefragments in tumor metastasis and the underlying mechanism, set toinvestigate how overexpression of full-length and the fragments ofADAMTS-1 affects metastasis of TA3 mammary carcinoma (TA3) cells. Asshown in FIGS. 1B and C, the C-terminal cleavage fragments of ADAMTS-1are heterogenic in their molecular weight, suggesting that ADAMTS-1 arecleaved at more than one sites within the spacer/Cys-rich region (FIG.1A, arrows). The molecular weight of the shortest C-terminal cleavagefragments is similar to that of the expressed C-terminal fragment ofADAMTS-1 containing the last two TSP-1 type I motifs (ADAMTS-1_(CTCF:)amino acids 842-951, FIG. 1A, D), suggesting that in addition to theprevious identified cleavage site in the spacer region (FIG. 1A, thebigger arrow), there is at least one additional cleavage site at thejunction between spacer region and the second TSP-1 type I motif (FIG.1A, the smaller arrow).

It was difficult to express the N-terminal fragments of ADAMTS-1containing different parts of the spacer and/or Cys-rich domains (datanot shown). In addition, studies have shown that auto-proteolyticcleavage of ADAMTS-4 occurs at the multiple sites within itsspacer/Cyr-rich region, and the shortest N-terminal cleavage fragment ofADAMTS-4 is generated by cleavage around the junction between theCys-rich domain and the TSP-1 type I motif. Thus, two expressionconstructs containing N-terminal fragments of ADAMTS-1 were made, whichexpressed well in TA3 cells. These constructs contain the N-terminaldomains of ADAMTS-1 until the end of the first TSP-1 type I motif(ADAMTS-1_(NTCF), amino acids 1-596, FIG. 1A, D) or until the end of thedisintegrin domain (ADAMTS-1_(minusTSP), amino acids 1-545, FIG. 1A, D).ADAMTS-1_(NTCF) likely represents the shortest N-terminal cleavagefragment of ADAMTS-1.

In order to assess the effects of ADAMTS-1 and its fragments on tumormetastasis reliably, the heterogeneity of TA3 cells was eliminated bytransfecting the cells with empty expression vector containingneomycin-resistant gene. A clonal TA3 cell (TA3_(wt1)) that undergoesaggressive pulmonary metastasis after intravenous (i.v.) injection wasselected (data not shown). Our RT-PCR result showed that like its wildtype counterpart, TA3_(wt1) cells express ADAMTS-1 endogenously (datanot shown). TA3_(wt1) was used to transfect several expressionconstructs that contain blasticidin-resistant gene and differentADAMTS-1 cDNA inserts (FIG. 1A). Five independent clonal TA3transfectants that were transfected with the empty expression vectoralone (TA3_(wtb)) or expressing the following gene products (FIG. 1D)were identified and used in pulmonary metastasis experiments: wild typeADAMTS-1 (TA3_(ADAMTS-1)), ADAMTS-1_(NTCF) (TA3_(ADAMTS-1NTCF)),ADAMTS-1_(CTCF) (TA3_(ADAMTS-1CTCF)), and ADAMTS-1_(minusTSP)(TA3_(ADAMTS-1minusTSP)). These TA3 transfectants displayed similargrowth rate in cell culture condition (data not shown).

Our results showed that overexpression of ADAMTS-1 significantlyaccelerated pulmonary metastasis and shortened the survival time of themice (FIG. 2A-C). On the contrary, ADAMTS-1_(NTCF) or ADAMTS-1_(CTCF),but not ADAMTS-1_(minusTSP) blocks pulmonary metastasis of the TA3transfectants (FIG. 2A-C), suggesting that the inhibitory effect ofthese ADAMTS-1 fragments is likely derived from the TSP type I motifswhich exist in ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF), but not inADAMTS-1_(minusTSP); and the anti-tumor activity is likely masked infull-length ADAMTS-1.

The metastatic tumors derived from TA3_(wtb), TA3_(ADAMTS-1), andTA3_(ADAMTS-1minusTSP) cells are invasive and fused together (FIGS. 2A,D, a-b, and data not shown), which made it difficult to determineaccurate number of the metastatic lesions. Thus, metastatic burden ofthe experimental mice was quantified by average weight of theexperimental mouse lungs (FIG. 2C). Because there is a significantdifference in survival time of these mice and the mice usually succumbto pulmonary metastasis when metastatic burden causes the lung weight toreach 1-1.2 grams, the metastatic burden of the remaining survival miceat day 11 and day 18 after i.v. injection of the TA3 transfectants wasmeasured. At least fifteen experimental mouse lungs were measured foreach type of the transfectants at each time point. Our results showedthat overexpression of ADAMTS-1 accelerated time that is need to reachthe maximal metastatic burden and shortened the survival time of themice, while overexpression of ADAMTS-1_(NTCF) or ADAMTS-1_(CTCF) blockedpulmonary metastasis and render most of the experimental mice free ofmetastatic disease (FIG. 2B-C).

Histologic analysis of the lung sections showed that TA3_(wtb),TA3_(ADAMTS-1), and TA3_(ADAMTS-1minusTSP) cells are invasive and fillup the pulmonary space (FIG. 2D-a-b, and data not shown). On thecontrary, only micrometastasis were detected in the lungs receivedTA3_(ADAMTS-1NTCF) or TA3_(ADAMTS-1CTCF) cells (FIG. 2D-c, d, arrows).To assess whether ADAMTS-1 expressed by the transfected TA3 cells iscleaved in vivo, different pulmonary tumors derived from TA3_(ADAMTS-1)cells were lysed and the proteins were analyzed by Western blotting withanti-v5 antibody, which recognizes the v5-tagged ADAMTS-1. The resultshowed that ADAMTS-1 protein is maintained in full-length form in vivoand no cleavage fragments of ADAMTS-1 were detected (FIG. 2E). Thisresult suggests that proteolytic cleavage of ADAMTS-1 regulates ADAMTS-1function and the cleavage status of ADAMTS-1 in vivo determine itseffect (stimulatory or inhibitory) on tumor metastasis.

ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) Blocks Pulmonary Metastasis byInhibiting Proliferation and Inducing Apoptosis of Tumor Cells, and byRepressing Tumor Angiogenesis

To determine the mechanism underlying the pro-tumor effect offull-length of ADAMTS-1 and the anti-tumor effect of ADAMTS-1_(NTCF) andADAMTS-1_(CTCF), proliferation and apoptosis rates of the tumor cellsand the extent of tumor angiogenesis during pulmonary metastasis of TA3transfectants were analyzed. A 5-Bromo-2′-deoxy-uridine (Brdu)incorporation assay and in situ detection of apoptotic cells on thesections derived from the experimental mouse lungs (six days after i.v.injection of TA3 transfectants) was performed. Results demonstrated thatexpression of ADAMTS-1_(NTCF) or ADAMTS-1_(CTCF), but not that ofADAMTS-1_(minusTSP), inhibits proliferation and promotes apoptosis ofthe tumor cells, and inhibits tumor angiogenesis; while overexpressionof full-length exogenous ADAMTS-1 on the top of endogenous ADAMTS-1 hasweak effect on tumor cell proliferation and apoptosis and promotes tumorangiogenesis in vivo (FIG. 3). These results imply that ADAMTS-1 plays arole in releasing/activating growth/survival/angiogenesis factors in themicroenvironments, while ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)blocks/interferes activities of the factors that promote tumor cellproliferation and survival and tumor angiogenesis.

ADAMTS-1 Promotes Extravasation of the Tumor Cells and Activation ofEGFR And ErbB-2 In Vivo, and Promotes Shedding of AR and HB-EGF

Activation of EGFR and ErbB-2 is known to promote proliferation andsurvival of breast carcinoma cells and is essential for progression ofbreast cancers. To determine whether ADAMTS-1 promotes activation ofEGFR and/or ErbB-2 in vivo, activity of EGFR and ErbB-2 in the lungswhere TA3_(wtb), TA3_(ADAMTS-1), TA3_(ADAMTS-1NTCF), orTA3_(ADAMTS-1CTCF) cells were injected intravenously 24 hours prior wasassessed. In order to normalizing amount of the tumor cells that wereincluded in the protein lysates and used in the immunoprecipitation, atumor cells tracking assay to determine the pulmonary extravasation rateof TA3 transfectants that were injected intravenously into theirsyngenic mice 24 hours prior was performed. The result showed thatoverexpression of ADAMTS-1 promotes tumor cell extravasation into lungparenchyma, while expression of TA3_(ADAMTS-1NTCF), orTA3_(ADAMTS-1CTCF) inhibits the process (FIG. 4A-B).

Normal mouse lungs and the mouse lungs that received TA3 transfectantsintravenously 24 hour prior were lysed, and the protein lysates thatstatistically contain the same amount of the tumor cells were used inimmunoprecipitation to pull-down EGFR or ErbB-2 and anti-phosphotyrosineantibody was used to detect phosphor-EGFR or phosphor-ErbB-2. The resultshowed that overexpression of ADAMTS-1 promotes activation of EGFR andErbB-2 (FIG. 4C). On the contrary, expression of ADAMTS-1_(NTCF) orADAMTS-1_(CTCF) blocks activation of EGFR and ErbB-2 (FIG. 4C).

Whether increased activation of EGFR and ErbB-2 induced by ADAMTS-1 isachieved via shedding/activating EGF family GF precursors, the ligandsof ErbB receptor tyrosine kinases which include EGFR, ErbB-2, -3, and-4. EGF family GFs include EGF, transforming growth factors-α (TGF-α),HB-EGF, AR, betacellulin, epiregulin, neuregulin, and epigen, and areshed from cell surface was investigated. Increasing amount of datasuggests that shedding of the EGF family GF precursors are essential inregulating availability and bioactivity of these factors and inactivation of the ErbB signaling pathways. The members of ADAM family,especially ADAM17 have been shown to play major but not sole role inshedding of these factors.

To determine whether ADAMTS-1 play an important role in constitutiveshedding EGF family GFs especially the ones that bind to heparin, Cos-7cells with several EGF family GF precursors that are expressed by TA3cells (data not shown) including HB-EGF, AR, and epigen with wereco-transfected with empty expression vector or the expression constructscontaining full-length ADAMTS-1, ADAMTS-1E/Q or various ADAMTS-1fragments. Serum-free cell culture medium (SFM) of the co-transfectedCos-7 cells were collected, concentrated and analyzed. Cos-7 cellsexpress endogenous ADAMTS-1 (data not shown). Overexpression ofexogenous ADAMTS-1 promotes shedding of AR and HB-EGF but not sheddingof epigen (FIG. 4D). More importantly, overexpress ADAMTS-1E/Q whichacts as a dominant negative regulator of endogenous ADAMTS-1 completelyblocks the shedding of AR and inhibits the shedding of HB-EGF, whileADAMTS-1 fragments displayed no significant effect on the shedding (FIG.4D). These data suggest that ADAMTS-1 promotes activation of EGFR andErbB-2 by promoting shedding and activation of the EGF family GFs.

ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) Inhibit Activation of Erk1/2 KinasesInduced by the EGF Family GFs

Since ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) display no significantinhibitory effect on shedding of AR and HB-EGF, it was investigated asto whether ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) inhibits activation ofEGFR and ErbB-2 by interfering activity of the soluble EGF family GFs.To assess that, purified soluble AR or HB-EGF was applied to MCF-10Amammary epithelial cells in the presence and absence of the naturalizingantibodies to HB-EGF or AR, ADAMTS-1_(NTCF), ADAMTS-1_(CTCF), orfull-length ADAMTS-1. This result showed that the neutralizingantibodies, ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF), but not full-lengthADAMTS-1 inhibit Erk1/2 kinase activation induced by soluble AR andHB-EGF (FIG. 5). This result suggests that ADAMTS-1_(NTCF) orADAMTS-1_(CTCF) inhibits activation of EGFR and ErbB-2 by inhibitingtheir ligand activity likely via interfering the binding between ligandsand their receptors and that the different effects of ADAMTS-1 and itscleavage fragments on availability and activity of soluble AR and HB-EGFunderlie their opposite roles in tumor metastasis.

To further determine the molecular mechanism underlying theanti-angiogenic activity of ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF), it wasinvestigated how these fragments affect activities of several importantgrowth/angiogenic factors that are known to regulate angiogenesis.Bioactivity of VEGF₁₆₅, basic FGF (bFGF), HB-EGF, and AR were revealedby their ability to induce activation of Erk1/2 kinases in HUVECs in thepresence or absence of purified ADAMTS-1 or the ADAMTS-1 fragments. Ourresults showed that ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) but notfull-length ADAMTS-1 or ADAMTS-1_(minusTSP) block activation of Erk1/2kinases induced by VEGF₁₆₅, HB-EGF, and AR but not that induced by bFGF(FIG. 6). These results suggest that ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)block tumor angiogenesis by sequestering the activities of severalimportant heparin binding factors that are essential for endothelialcells proliferation and survival.

ADAMTS-1 was found to inhibit tumor growth by blocking tumorangiogenesis; however, this study did not investigate whether theanti-tumor activity is derived from the full-length ADAMTS-1, itscleavage fragments, or both. On the contrary, increased expression ofADAMTS-1 was correlated to the increased metastatic potential in theclinic tumor samples. The current study was designed to betterunderstand the role of full-length and the cleavage fragments ofADAMTS-1 in tumor metastasis and to elucidate the underlying mechanisms.It was demonstrated that overexpression of ADAMTS-1 promotes tumormetastasis by promoting tumor cell extravasation and tumor angiogenesis.It is well established that tumor cell extravasation is a critical stepduring tumor metastasis and studies have shown that ADAMTS-1 is capableof degrading aggrecan and versican. The ability of ADAMTS-1 to degradeaggrecan/versican and other not yet identified ECM components is likelyresponsible for the enhanced extravasation ability of TA3_(ADAMTS-1)cells. Furthermore, as described herein, ADAMTS-1 promotes shedding ofAR and HB-EGF, which in turn promotes activation of EGFR and ErbB-2 andproliferation and survival of the tumor cells in vivo.

In the current study, it was demonstrated that ADAMTS-1 undergoesauto-proteolytic cleavage and overexpression of the cleavage fragmentsof ADAMTS-1 (ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)) block metastasis ofTA3 cells by inhibiting extravasation, proliferation and survival of thetumor cells, and by repressing tumor angiogenesis via interferingactivities of several important heparin binding growth/angiogenicfactors. Furthermore, it was demonstrated that auto-proteolytic cleavageof ADAMTS-1 is blocked by HS, which suggests that the level of HS/HSPGin the microenvironment likely regulates which form of ADAMTS-1(full-length or the cleavage fragments) presents predominantly in themicroenvironment to exert pro- or anti-tumor activity, respectively.Thus, the roles of ADAMTS-1 and its cleavage fragments in tumormetastasis, provided the regulatory mechanism of ADAMTS-1 function (byauto-proteolytic cleavage and HS/HSPGs), and revealed the mechanismsunderlying the function of ADAMTS-1 and the ADAMTS-1 cleavage fragments(by regulating availability and activity of the EGF family GFs and ErbBsignaling pathway).

Shedding EGF Family GFs by ADAMTS-1

Although functional differences between mature soluble EGF family GFsand their transmembrane precursors are not well-established, thephenotype similarity between TGF-α- and ADAM17-null mice and betweenHB-EGF-null and HB-EGF cleavage resistant mice clearly suggested thatshedding of these precursors is essential for availability and activityof these factors. Several members of ADAM family including ADAM 9, 10,12, 17 have been implicated in shedding of HB-EGF and AR. The studiesusing the cells derived from ADAM-9, -10, -12, -15, and/or -17 null-micehave suggested that ADAM17 are the major but not the sole sheddase of ARand HB-EGF, and other member(s) of ADAM and/or ADAMTS family is(are)likely play important roles as well, especially in thenon-PMA-induced/metalloproteinase inhibitor sensitive/constitutiveshedding of these factors.

Several members of EGF family GFs including HB-EGF and AR bind toHS/HSPGs. ADAMTS-1 binds to HS as well through the spacer region and theTSP type I motifs, which brings the proteinase domain of ADAMTS-1 closeto the HS/HSPG bound factors and makes ADAMTS-1 as an ideal sheddase tocleave these HS/HSPG binding GF precursors. The present disclosure hasprovided evidences that ADAMTS-1 promotes shedding of AR and HB-EGF andADAMTS-1 may be a major sheddase that is responsible for constitutiveshedding of AR and HB-EGF. Soluble AR and HB-EGF shed by ADADMTS-1 canin turn promote tumor cell survival and proliferation and tumorangiogenesis in vivo.

As discussed herein, it is shown that ADAMTS-1 but not the ADAMTS-1fragments promotes shedding of AR and HB-EGF, suggesting that the intactspacer/Cys-rich domain is required for the shedding and thespacer/Cys-rich domain contains substrate recognition/binding site(s)which is(are) destroyed by the auto-proteolytic cleavage in this region.Since all the members of ADAMTS family have similar domain organization,in addition to ADAMTS-1, other members of the ADAMTS family may alsoinvolve in regulating availability and activity of HS/HSPG-bindingfactors.

The Anti-Tumor Activity of the ADAMTS-1 Fragments is Masked in theFull-Length Molecule.

As described herein, it is demonstrated that in contrast to the effectof full-length ADAMTS-1, the ADAMTS-1 cleavage fragments(ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)) block pulmonary metastasis of TA3cells. How can auto-proteolytic cleavage convert a pro-tumor factor intoanti-tumor ones? The results suggest that auto-proteolytic cleavagedestroys the substrate binding domain in the spacer/Cys-rich region thatis likely required for binding to AR and HB-EGF precursors. In addition,it is described herein that the N-terminal deletion fragment of ADAMTS-1in which all the TSP type I motifs were deleted (ADAMTS-1_(minusTSP))displayed no anti-tumor activity, suggesting that the anti-tumoractivity of ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) is derived from the TSPtype I motif. Even though full-length ADAMTS-1 contains all three TSPtype I motifs, they are likely masked and unable to exert anti-tumoractivity. Auto-proteolytic cleavage of ADAMTS-1 at the spacer/Cys-richregion not only renders the N-terminal cleavage fragment(ADAMTS-1_(NTCF)) that contains the metalloproteinase domain incapableof binding to and shedding AR and HB-EGF precursors (FIG. 4D), but alsoexposes the cryptic anti-tumor domains in both N- and C-terminalcleavage fragments. In addition to ADAMTS-1, ADAMTS-4, and -12 undergoproteolytic cleavage at their spacer/Cys-rich region as well. Theauto-proteolytic cleavage may be a general mechanism that regulatesfunction of many ADAMTS family members, and our results provided thefirst example of this type of regulatory mechanism.

As described herein, it has been shown that ADAMTS-1_(NTCF) andADAMTS-1_(CTCF) inhibits activation of Erk1/2 kinase induced by AR,HB-EGF, or VEGF (FIG. 5-6). A recent study has shown that ADAMTS-1inhibits VEGF activity by blocking the binding between VEGF and theirreceptor. Although additional study is required to determine the exactbiochemical mechanism underlying the inhibitory effect ofADAMTS-1_(NTCF) and ADAMTS-1_(CTCF), they likely exert their inhibitoryeffect by sequestering these soluble GFs from their receptors.

The Function of ADAMTS-1 is Regulated by HS/HSPGs

As described herein, heparin/HS blocks auto-proteolytic cleavage ofADAMTS-1, and full-length ADAMTS-1 and the ADAMTS-1 cleavage fragments(ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)) displayed opposite effects ontumor metastasis. Thus, ability of ADAMTS-1 to inhibit or promote tumormetastasis is dependent on the ability of tumor cells and theirsurrounding microenvironment to cleave ADAMTS-1. In other words, in amicroenvironment that is highly enriched HS and HSPGs, binding ofADAMTS-1 to HS/HSPGs protects the proteolytic cleavage sites in thespacer/Cys-rich region which keeps ADAMTS-1 in the full-length form andin turn binds and cleaves its substrates including transmembrane AR andHB-EGF. In that situation, full-length ADAMTS-1 exerts pro-tumoractivity by releasing and activating pro-proliferation, -survival, and-angiogenic factors. In addition, the anti-tumor activity derived fromTSP type I motifs is likely masked in full-length ADAMTS-1. On thecontrary, in a microenvironment that is lack of or low in HS/HSPGs,ADAMTS-1 is likely cleaved to generate the cleavage fragments that arewithout the substrate (AR and HB-EGF) binding site(s) and contain unmaskthe anti-tumor TSP type I motifs.

Example 3 The Pro-Tumor Effect of Full-Length ADAMTS-1 and theAnti-Tumor Effect of the ADAMTS-1 Fragments were Confirmed in Lewis LungCarcinoma (LLC) Cells

To confirm the effects of full-length ADAMTS-1 and the ADAMTS-1fragments on tumor growth and metastasis, and compare their effects withthat of thrombospondin-1 and -2, LLC transfectants were established thatwere transfected with empty expression vectors (LLC_(wtb)) or expressingfull-length ADAMTS-1, ADAMTS-1E/Q, ADAMTS-1_(NTF), ADAMTS-1_(CTF),thrombospondin-1, or -2 (FIG. 16). ADAMTS-1, thrombospondin-1 and -2 arethe members of thrombospondin type I repeat superfamily (TRS).Thrombospondin-1 is a 450 kDa homotrimeric ECM protein and is consideredas a potent anti-tumor molecule. Studies have shown that systemicinjection or overexpression of thrombospondin-1 inhibits the in vivogrowth of several tumor cells including LLC cells (70, 78, 79). Asubline of LLC cell (LLC_(wt)) that undergoes spontaneous pulmonarymetastasis after removal of the primary subcutaneous (s.c.) tumors wereused to establish these transfectants. LLC_(wt) cells express a lowlevel of endogenous ADAMTS-1 as assessed by RT-PCR and Western blotanalysis (data not shown). Five independent clonal LLC transfectantsexpressing a high to intermediate level of the same gene products wererandomly selected, pooled (FIG. 16), and used in the s.c. tumor growthand spontaneous pulmonary metastasis experiments following theestablished protocols (80-82, 108, 110).

The results showed that expression of full-length ADAMTS-1 promoteswhile expression ADAMTS-1_(NTF) or ADAMTS-1_(CTF) and to a less extentthat of ADAMTS-1E/Q inhibits s.c. growth and spontaneous pulmonarymetastasis of the LLC transfectants (FIG. 16). More importantly, eventhough the LLC transfectants express a higher level of thrombospondin-1or -2 comparing to that of ADAMTS-1NTF and ADAMTS-1CTF, the inhibitoryeffect derived from the ADAMTS-1 fragments is stronger than that derivedfrom thrombospondin-1 or -2 (FIG. 16), suggesting that the ADAMTS-1fragments and their derivatives have unique features and a greatpotential to be used as the potent anti-cancer agents.

The Metalloproteinase Activity in ADAMTS-1_(NTF) is not Required for itsAnti-Tumor Activity:

To investigate whether metalloproteinase activity in ADAMTS-1_(NTF) isrequired for the anti-tumor activity of ADAMTS-1_(NTF), a protease-deadADAMTS-1NTFE/Q mutant was established, in which E386 is switched to Q inthe Zinc-binding pocket of the metalloproteinase domain. The expressionconstructs were used to transfect TA3 mouse mammary carcinoma cells.Three independent positive colonies that express ADAMTS-1NTFE/Q orADAMTS-1NTF or transfected with the empty expression vectors (FIG. 17)were used in the pulmonary tumor metastasis experiments. Our resultsshowed that ADAMTS-1_(NTF)E/Q behaved like ADAMTS-1_(NTF) andsignificantly promoted the survival of the experimental mice andinhibited the pulmonary tumor metastasis (FIG. 17). This result suggeststhat the metalloproteinase domain of ADAMTS-1 does not contribute to theanti-tumor effect of ADAMTS-1_(NTF).

Example 4

The anti-tumor and anti-angiogenic activity of thrombospondin-1 has beenwell established and the anti-tumor activity has been mapped to theseveral domains including the TSP type I repeats. All the members of theADAMTS family contain at least one TSP-1 motif and belong to thethrombospondin type I repeat (TSR) superfamily (73). Sinceidentification of ADAMTS-1 (22), several studies have been performed toinvestigate the role of ADAMTS-1 in tumor growth and metastasis, and theresults obtained appeared to contradict each other. In pancreatic cancersamples, a higher ADAMTS-1 mRNA level was correlate to the severe lymphnode metastasis or retroperitoneal invasion and worse prognosis,suggesting that ADAMST-1 likely promotes pancreatic cancer invasion andmetastasis. However, ADAMTS-1 mRNA is down-regulated in the breastcarcinoma samples comparing to the nonneoplastic mammary tissues butwith no strong links between the ADAMTS-1 mRNA level and theclinicopathological features of these breast cancer cases studied. Thesestudies have only measured ADAMTS-1 mRNA level but not the protein leveland the proteolytic activity of ADAMTS-1, both of which are morerelevant to the ADAMTS-1 function.

In addition, ADAMTS-1 was found to inhibit tumor growth by blockingtumor angiogenesis, which is likely achieved by sequestering VEGF₁₆₅from its receptor, and the metalloproteinase activity of ADAMTS-1 isrequired for the observed anti-angiogenesis and anti-tumor growthactivity. In contrast to this finding, overexpression of ADAMTS-1 wasfound to promote subcutaneous growth of the transfected CHO cells butinhibit experimental metastasis of the same transfectants. However,these studies have neither considered the fact that ADAMTS-1 isproteolytically cleaved, nor investigated the cleavage status ofADAMTS-1 in vivo (in subcutaneous and pulmonary microenvironments), anddid not consider the possibility that the requirement of themetalloproteinase activity of ADAMTS-1 for its anti-tumor effect maymerely reflect to the fact that the anti-tumor effect is actuallyderived from the auto-proteolytic cleavage fragments but not thefull-length ADAMTS-1, and that the metalloproteinase activity ofADAMTS-1 is required for generating these ADAMTS-1 fragments.

To test this possibility, the how and why full-length and the ADAMTS-1fragments affect tumor growth and metastasis was investigated. Asdescribed herein, it is demonstrated that overexpression of full-lengthADAMTS-1, which is maintained in the full-length form during metastasisof TA3 mammary carcinoma cells, promotes the tumor metastasis, and thatADAMTS-1 promotes shedding of AR and HB-EGF precursors and activation ofEGFR and ErbB-2 in vivo. In addition, for the first time that ADAMTS-1undergoes auto-proteolytic cleavage to generate the NH₂- andCOOH-terminal fragments that contain at least one TSP-1 motif is shown.In contrast to that of full-length ADAMTS-1, overexpression of thefragments of ADAMTS-1 (ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF)) that mimicthe proteolytic cleavage fragments of ADAMTS-1 blocks pulmonarymetastasis of TA3 cells by inhibiting tumor cell extravasation,proliferation and survival, and by repressing tumor angiogenesis. It isdemonstrated that the anti-metastatic activity of the ADAMTS-1 fragmentsrequires the TSP-1 motif, which is likely masked in the full-lengthmolecule, and that ADAMTS-1_(NTF) and ADAMTS-1_(CTF) inhibit activationof EGFR and ErbB-2 in vivo and inhibits the Erk1/2 kinase activationinduced by soluble AR and HB-EGF.

Furthermore, it is demonstrated that the proteolytic cleavage ofADAMTS-1 is blocked by heparin, and HS, suggesting that the binding ofADAMTS-1 to heparan sulfate proteoglycans (HSPGs) masks theauto-proteolytic cleavage site(s) in the spacer/Cys-rich domain and keepADAMTS-1 in the full-length form to cleave their substrates. On theother hand, the auto-proteolytic cleavage of ADAMTS-1 in thespacer/Cys-rich domain likely destroys the substrate binding sites andunmasks the anti-tumor TSP-1 domain, which renders the anti-tumoractivity to the ADAMTS-1 fragments. Thus, the level of HSPGs in themicroenvironment likely regulates the form of ADAMTS-1 (full-length orthe cleavage fragments) that exists predominantly in themicroenvironment to exert pro- or anti-tumor activity, respectively. Itis demonstrated that ADAMTS-1 expressed by TA3 cells is maintained inthe full-length form in vivo to exert pro-metastasis activity. Thus, theresults have reconciled the apparent contradiction in the previousresults and demonstrated that the cleavage status of ADAMTS-1 determinesits effect (stimulatory or inhibitory) on tumor growth and metastasis.

The results described herein suggested that ADAMTS-1 plays the multipleroles in tumor growth and metastasis and is a prime target for cancertherapy, and that the ADAMTS-1 fragments have great potential as thepotent anti-cancer agents that inhibit not only tumor cellproliferation/survival/invasion, but also tumor angiogenesis.

The Pro-Metastatic Activity of Full-Length ADAMTS-1 Requires itsMetalloproteinase Activity

To determine whether the metalloproteinase activity of ADAMTS-1 isrequired for the pro-metastatic activity of ADAMTS-1, TA3 transfectantsexpressing the protease-dead mutant of ADAMTS-1 (ADAMTS-1E/Q), whichharbors an E₃₈₆ to Q point mutation in the Zinc-binding pocket of themetalloproteinase domain were generated. The study has shown that thismutant lacks the catalytic activity.

In order to assess the effects of ADAMTS-1E/Q on tumor metastasisreliably, the established clonal TA3 cell line, TA3_(wt1) was used. Likeits parental cells, TA3_(wt1) cells express ADAMTS-1 endogenously andundergo pulmonary metastasis after intravenous (i.v.) injection. Fiveindependent clonal TA3 transfectants expressing a high to intermediatelevel of ADAMTS-1E/Q were randomly selected and used as the pooledpopulation (TA3_(ADAMTS-1E/Q), FIG. 7B) in the pulmonary metastasisexperiments. Five independent clonal TA3 transfectants transfected withthe empty expression vectors or expressing the following same geneproducts were used as the pooled population as well: full-lengthADAMTS-1 (TA3_(ADAMTS-1)), ADAMTS-1_(NTF) (TA3_(ADAMTS-1NTF)),ADAMTS-1_(CTF) (TA3_(ADAMTS-1CTF)), and ADAMTS-1_(minusTSP-1)(TA3_(ADAMTS-1minusTSP-1)). These pooled TA3 transfectants express asimilar level of the transfected gene products (FIG. 7B) and displayed asimilar growth rate in the cell culture condition with 10% FBS (data notshown).

It was confirmed that the expression of full-length ADAMTS-1 promotesthe pulmonary metastasis of TA3 cells and shortens the survival time ofthe mice, while ADAMTS-1_(NTF) or ADAMTS-1_(CTF), but notADAMTS-1_(minusTSP-1) blocks the pulmonary metastasis of thetransfectants (FIG. 7C-D). In addition, the expression of ADAMTS-1E/Qinhibits the pulmonary metastasis of the transfectants and extends thesurvival time of the mice (FIG. 7C-D), suggesting that themetalloproteinase activity is required for the pro-metastatic activityof full-length ADAMTS-1. The metastatic burden was quantified by theaverage weight of the experimental mouse lungs (FIG. 7D). Because thereis a significant difference in the survival time of the experimentalmice which succumb to pulmonary metastasis when metastatic burden causesthe lung weight to reach 1-1.2 gram, the metastatic burden was measuredin the remaining survival mice at day 12 and 20 after i.v. injection ofthese TA3 transfectants. At least 12 mouse lungs were weighted for eachtype of the transfectants at each time point. We confirmed thatoverexpression of full-length ADAMTS-1 accelerated the time that isrequired to reach the maximal metastatic burden and shortened thesurvival time of the mice, while overexpression of ADAMTS-1E/Q,ADAMTS-1_(NTF), or ADAMTS-1_(CTF) but not ADAMTS-1_(minusTSP-1) reducedthe metastatic burden (FIG. 7D). Furthermore, it was demonstrated thatthe inhibitory effect derived from ADAMTS-1_(NTF) or ADAMTS-1_(CTF) isstronger than that derived from ADAMTS-1E/Q, implying that theunderlying mechanisms for their anti-metastatic effects may bedifferent. This hypothesis was supported by the results obtainedpreviously, which indicated that ADAMTS-1E/Q but not ADAMTS-1_(NTF) andADAMTS-1_(CTF) serves as a dominant negative regulator of full-lengthendogenous ADAMTS-1 by inhibiting the shedding of HB-EGF and ARtransmembrane precursors (FIG. 4). Together, these data suggest thatlike full-length ADAMTS-1, the anti-tumor TSP-1 domains in ADAMTS-1E/Qare masked, and that the anti-tumor activity is likely derived from theintact spacer/Cys-rich domain, which competes with ADAMTS-1 for thebinding to its substrates.

The Spacer/Cys-Rich Domain is Essential for Binding of ADAMTS-1 to theCell Surface and the ECM

The ADAMTS-1 substrates identified so far are versican, aggrecan, andHB-EGF and AR precursors, which are located on the ECM and the cellsurface, respectively. To determine the domain(s) of ADAMTS-1 thatmediate(s) the substrate binding, we first assess the ECM and the cellbinding capacity of the different deletional mutants of ADAMTS-1 (seeFIG. 7A). All the constructs contain the COOH-terminal v5-epitope tagsfor easy identification and purification, and the constructs weretransfected into COS-7 cells. 72 hours after the transfection, theproteins derived from the cell culture supernatants, the ECM materialsdeposited by the transfected cells, and the lysates of the transfectedcells were analyzed by Western blotting with anti-v5 epitope antibody asdescribed (106, 109). The results showed that the spacer/Cys-rich domainis essential for the binding of ADAMTS-1 to the ECM and the cells (FIG.8), suggesting that the spacer/Cys-rich domain likely mediates thesubstrate binding of ADAMTS-1.

ADAMTS-1 Promotes Invasion of TA3 Cells Through Matrigel

It is well established that the pericellular proteolysis mediated byMMPs is essential for tumor invasion. As a member of theZinc²⁺-dependent metalloproteinase family, ADAMTS-1 plays an importantrole in degrading versican, an important component of the ECM and theblood vessel walls. We have shown that ADAMTS-1 promotes extravasationof TA3 cells into lung parenchyma (FIG. 4). To determine how full-lengthADAMTS-1 and the fragments of ADAMTS-1 affect tumor cell invasionthrough Matrigel which mimics the basement membrane as the barriers oftumor cell invasion, an invasion assay by using Transwell cell culturechambers with 8-μm pores (Costar) coated with a layer of Matrigel(Collaborative Biomedical) was performed. The DMEM containing 2% FBS wasbe added into the lower chambers of the Transwells. 2×10⁵ of thedifferent TA3 transfectants were seeded on top of the Transwell intriplicate and incubated for 24 hours. The bottom filters were then befixed and stained. The cells on the top chambers were removed by wipingwith cotton swabs, and the stained cells (blue color) that have migratedthrough the Matrigel were counted under a microscope. Six randomlyselected 100× microscopic fields will be countered. The invasion indexof the different TA3 transfectants was calculated as following formula:

${{Invasion}\mspace{14mu} {Index}} = {100\% \times \frac{{Average}\mspace{14mu} {numbers}\mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {in}\mspace{14mu} {lower}\mspace{14mu} {{camber}/{microscopic}}\mspace{14mu} {field}}{{Numbers}\mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {seeded}\mspace{14mu} {on}\mspace{20mu} {upper}\mspace{14mu} {{camber}/{microscopic}}\mspace{14mu} {field}}}$

The results showed that TA3_(ADAMTS-1) cells displayed approximately twotime higher invasion index than TA3_(wt1) and TA3_(ADAMTS-1minusTSP-1)cells, and four-eight time higher invasion index compared toTA3_(ADAMTS-1NTF)/TA3_(ADAMTS-1CTF) and TA3_(ADAMTS-1E/Q) cells,respectively (FIG. 9A-B). These results further confirmed that ADAMTS-1promotes tumor cell invasion, while TA3_(ADAMTS-1E/Q) and to a lessextent TA3_(ADAMTS-1NTF), or TA3_(ADAMTS-1CTF) inhibits the process. Todetermine whether ADAMTS-1 promotes TA3 cell invasion by degradingversican or inhibiting the pro-migratory effect of soluble HB-EGF andAR, the confluence TA3 transfectants were lifted by the EDTA solutionand the ECM materials remained on the cell culture dishes were extractedand analyzed by Western blotting with anti-DP antibody, which detectsthe cleavage fragments of versican. The result showed that increasedexpression of exogenous ADAMTS-1 but not ADAMTS-1_(minusTSP-1) on top ofthe endogenous ADAMTS-1 promotes degradation of versican, whileexpression of ADAMTS-1E/Q but not ADAMTS-1_(NTF), ADAMTS-1_(CTF)inhibits the degradation (FIG. 9C). These data suggest that ADAMTS-1E/Qinhibits TA3 cell invasion by blocking the ADAMTS-1 mediated versicandegradation, while the weaker inhibitory effect of the ADAMTS-1fragments is likely derived from their indirect effect on activity ofHB-EGF/AR.

Example 5

Following the experimental procedures described in Example 1, additiondata was generated showing additional differences between full lengthADAMTS-1 and its cleavage products, ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF).Overexpression of ADAMTS-1 promotes growth of TA3 mammary carcinoma(TA3) cells while overexpression of the N- or C-terminal fragment ofADAMTS-1 blocks growth of TA3 cells by inhibiting proliferation andinducing apoptosis of the tumor cells and by inhibiting tumorangiogenesis. ADAMTS-1 expressed by TA3 cells maintained in thefull-length form in vivo exerted pro-tumor growth and metastasisactivity. In contrast to the of full-length ADAMTS-1, overexpression ofthe N- or C-terminal fragment of ADAMTS-1 (ADAMTS-1_(NTCF) andADAMTS-1_(CTCF)) inhibits subcutaneous (s.c.) growth of TA3 cells. Inaddition, unlike full-length ADAMTS-1 which promotes shedding of the EGFfamily ligands including amphiregulin (AR) and heparin-binding EGH(HB-EGF) and activation of EGF receptor (EGFR) and ErbB-2, the ADAMTS-1fragments inhibits activation of EGFR and ErbB-2 in vivo.

RT-PCR results showed that like wild type TA3 cells, TA3_(wt1) cellsexpress ADAMTS-1 endogenously as do several other tumor cell lines (FIG.10). Growth rates of the s.c. solid tumors derived from different TA3transfectants were measured and the result showed that overexpression ofADAMTS-1 promotes tumor growth, while overexpression of ADAMTS-1_(NTCF)and ADAMTS-1_(CTCF), but not that of ADAMTS-1_(minusTSP) significantlyinhibits tumor growth (FIG. 11). These results suggest that theinhibitory effect of the ADAMTS-1 fragments is likely derived from theTSP type I motifs, which exist in ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF),but not in ADAMTS-1_(minusTSP). The data show that ADAMTS-1_(NTCF) orADAMTS-1_(CTCF), blocks pulmonary metastasis of TA3ADAMTS-1_(NTCF) orTA3ADAMTS-1_(CTCF) cells (FIG. 11, B). The metastatic burden of theexperimental mice was quantified by average weight of the experimentalmouse lungs received different TA3 transfectants (FIG. 11, B). Resultsshowed that overexpression of ADAMTS-1_(NTCF) or ADAMTS-1_(CTCF)dramatically reduced metastatic burden of the mice received thecorresponding TA3 transfectants, and render most of the experimentalmice free of metastatic disease and significantly extended survival timeof these mice (FIG. 11, B). [0180] The ADAMTS-1 fragments blocks tumorgrowth by inhibiting proliferating and inducing apoptosis of tumorcells, and inhibiting tumor angiogenesis. To determine the cellularbasis of the pro-tumor effect of full-length of ADAMTS-1 and theanti-tumor effect of ADAMTS-1 fragments, proliferation and apoptosisrates of the tumor cells and tumor angiogenesis during s.c. growth wereanalyzed. Brdu (5-Bomo-2′-deoxy-uridine) incorporation assay and in situdetection of apoptotic cells were performed on the sections derived froms.c. solid tumors (twelve days after implanting the TA3 cells). Resultsdemonstrated that expression of ADAMTS-1_(NTCF) and to a less extentthat of ADAMTS-1_(CFCF), but not expression of ADAMTS-1_(minusTSP),inhibits proliferation and promotes apoptosis of the tumor cells, andinhibits angiogenesis in the subcutaneous space; while expression ofexogenous ADAMTS-1 mildly enhances proliferation rate and reducesapoptosis rate of the tumor cells, and promotes tumor angiogenesis invivo (FIG. 12) These results suggest that ADAMTS-1 may play an importantrole in releasing/activating growth/survival factors in themicroenvironments, while the cleavage fragments of ADAMTS-1 may blockthe activities of the factors that promote tumor cell proliferation andsurvival and tumor angiogenesis.

Activation of EGFR and ErbB-2 is known to promote proliferation andsurvival of breast carcinoma cells and play essential roles inprogression of breast cancers. To determine whether activation of EGFRand/or ErbB-2 underlies the pro-tumor activity of ADAMTS-1, we assessedactivity of EGFR and ErbB-2 in the lungs where TA3_(wtb),TA3_(ADAMTS-1), TA3ADAMTS-1_(NTCF), TA3ADAMTS-1_(NTCF), orADAMTS-1_(minusTSP) cells were injected five days prior. The resultshowed that expression of ADAMTS-1 by TA3 cells promotes activation ofEGFR and ErbB-2 in vivo (FIG. 13, A). On the contrary, expression ofADAMTS-1_(NTCF) or ADAMTS-1_(CTCF), but not ADAMTS-1_(minusTSP) whichlacks TSP type I motifs, blocks activation of EGFR and ErbB-2 in vivo(FIG. 13, A).

Experiments were done to assess whether induces activation of EGFR andErbB-2 by ADAMTS-1 is achieved via promoting shedding EGF familyligands. EGF family ligands are produced as transmembrane precursors,which are shed and released from cell surface as soluble mature form.Several EGF family ligands are known to be shed-activated by the ADAMfamily proteinases including ADAM-17. However, studies using the cellsderived from ADAM-17 null-mouse suggested that ADAM-17 is not the soleproteinase that is responsible for shedding of TGF-α and other member(s)of ADAM family is (are) likely to play a role as well. To determinewhether ADAMTS-1 play a role in shedding EGF family ligands especiallythe ones that bind to heparin, several EGF family ligands wereco-transfected including HB-EGF, TGF-α, AR, and epigen which areexpressed by TA3 cells (data not shown) with or without the full-lengthADAMTS-1, ADAMTS-1E/Q and the ADAMTS-1 fragments. The serum-free cellculture medium of the transfected cells were collected and concentratedand analyzed. The results showed that ADAMTS-1 promotes shedding of ARand HB-EGF but not shedding of TGF-α and epigen; while ADAMTS-1E/Qblocks the shedding. The ADAMTS-1 fragments displayed no effect on theshedding (FIG. 13, B and data not shown).

To determine whether the ADAMTS-1 fragments affect the signaltransduction pathways activated by HB-EGF and AR, the serum-free cellculture media (SFM) derived from the co-transfected cells were appliedto MCF-10A mammary epithelial cells to determine their ability to induceErk1/2 kinase activation. The results showed that soluble HB-EGF and ARin the SFM induces activation of Erk1/2 kinases, which is specificallyblocked by the corresponding blocking antibodies or the ADAMTS-1fragments, but not by the full-length ADAMTS-1 (FIG. 13, C). This resultsuggests that the ADAMTS-1 fragments inhibit activation of EGFR andErbB-2 by interfering with their ligand activity; and the effects ofADAMTS-1 and its cleavage fragments on the availability and activity ofEGF family ligands likely underlies their roles in tumor growth andmetastasis.

The affect the fragments have on activities of several importantgrowth/angiogenic factors that are known to regulate angiogenesis wasinvestigated. Activity of VEGF₁₆₅, bFGF, HB-EGF, TGF-α, and AR wererevealed by their ability to induce activation of Erk1/2 kinases inHUVECs in the presence or absence of different purified ADAMTS-1proteins. Results showed that ADAMTS-1_(NTCF) and ADAMTS-1_(CTCF) butnot full-length ADAMTS-1 or ADAMTS-1_(minusTSP) block activation ofErk1/2 kinase induced by VEGF₁₆₅, TGF-α, HB-EGF, and AR

ADAMTS-1 is widely expressed by tumor cells and undergoesauto-proteolytic cleavage. In addition, overexpression of ADAMTS-1promotes tumor growth and metastasis by enhancing tumor cellproliferation and survival and by promoting tumor angiogenesis throughshedding transmembrane EGF family ligands, AR and HB-EGF, which in turnpromotes activation of EGFR and ErbB-2 in vivo.

The results not only provided a potential important target (full-lengthADAMTS-1), potent novel anti-cancer reagents (the ADAMTS-1 fragments),and the regulatory reagents for ADAMTS-1 activity (HS/HSPGs) for thetreatment of cancers especially breast cancers in the figure, but alsorevealed the mechanism underlying the function of ADAMTS-1 and theADAMTS-1 fragments.

The presence of TSP type I motif is a common feature of all members ofADAMTS family, among them ADAMTS-1, -4, AND -12 undergo proteolyticcleavage at their spacer/Cys-rich region, which have potential togenerate ADAMTS fragments containing unmasked TSP type I motifs that maypossess anti-tumor activity. In addition, as indicated in this study,the auto-proteolytic cleavage may be a general mechanism that regulatesthe function of ADAMTS family members. Additional work is required toverify these hypotheses and the results obtained in this study providegeneral rules that may apply the other ADAMTS family members as well.

For subcutaneous tumor growth experiments, five independent clonal TA3transfectants expressing ADAMTS-1, ADAMTS-1_(CTCF), ADAMTS-1_(NTCF) orADAMTS-1_(minusTSP), or transfected with the empty expression vectorwere used in the in vivo experiments. For each type of the experiment,six mice were injected with each clonal transfectants and twoindependent experiments were performed.

In tumor growth experiments, TA3 transfectants were injectedsubcutaneously into syngenic A/Jax-mic as described. After solid tumorsbecame visible (7-10 days after the injection), the tumors were measuredby a digital caliper every other day for the next two weeks. The largestand shortest diameters of the solid tumors were measured. The tumorvolume was calculated by using the following formula: tumorvolume=1/2×(shortest diameter)²×longest diameter (mm³)

Example 6 Determining the Exact Amino Acid Segments in the TSP-1 DomainsContaining Anti-Cancer Activity

As discussed herein, the ADAMTS-1 fragments that contain either themiddle TSP-1 motif (ADAMTS-1_(NTF)) or the two COOH-terminal TSP-1modules (ADAMTS-1_(CTF)) inhibit growth and/or metastasis of TA3 andLLC, and their inhibitory effect is much stronger than that caused bythrombospondin-1 and -2, suggesting the unique molecular basisunderlying the potent inhibitory effect of the ADAMTS-1 fragments is notpresent in thrombospondin-1 and -2. Also, the TSP-1 domain is requiredfor the anti-tumor activity of ADAMTS-1_(NTF). The middle TSP-1 domain(mTSP-1, amino acids 546-596) in ADAMTS-1 is similar but not identicalto the second and third TSP-1 repeats (WXXWXXW) in thrombospondin-1,which have been shown to contain anti-tumor and anti-angiogenic activity(102, 111-113). Even though the COOH-terminal TSP-1 modules (cTSP-1,amino acid 842-895 and 896-951, FIG. 9) of ADAMTS-1 do not have highhomology to the TSP-1 repeats in thrombospondin-1, the ADAMST-1fragments that contain either the mTSP-1 or cTSP-1 domain exhibited thesimilar anti-tumor activity, implying that the common unidentifiedunique amino acid segments or three dimension feature (other than theWXXWXXW) in the m/cTSP-1 domains of ADAMTS-1 may be essential for thepotent anti-tumor activity. Accordingly, the molecular basis for thepotent anti-tumor activity that is unique to the ADAMTS-1 fragments canbe identified using this domain. To achieve that, deletions in the TSP-1domains of ADAMTS-1 can be made and tumor growth and metastasis assayscan be performed using TA3 and LLC transfectants expressing theseADAMTS-1 mutants (as described herein).

To Determine Whether the _(m and/or c) fTSP-1 Domain Displays Anti-TumorActivity

It was shown that deletion of the middle TSP-1 (mTSP-1) domain fromADAMTS-1_(NTF) (ADAMTS-1_(minusTSP-1)) abolishes the potent anti-tumoractivity of the fragment, suggesting the anti-tumor activity resided inthe mTSP-1 domain and that the ADAMTS-1_(CTF) is composed of the twoCOOH-terminal TSP-1 (cTSP-1) domains. Thus, whether the mTSP-1 domainand each of the cTSP-1 domains display as potent anti-tumor activity asthe ADAMTS-1_(NTF) and ADAMTS-1_(NTF) fragments need to be determined.To achieve that, three expression constructs can be generated thatcontain the signal peptide plus the mTSP-1 domain (fADAMTS-1_(m-TSP-1)),the first cTSP-1 (fADAMTS-1_(cTSP-1-1)), or the second cTSP-1(fADAMTS-1_(cTSP-1-2)) domain in pEF/6/v5-His expression vectors. Theycan be used to transfect TA3wt1 and LLCwt1 cells. Five independent TA3or LLC transfectants expressing a high to intermediate level offADAMTS-1 mTSP-1, fADAMTS-1cTSP-1-1 or fADAMTS-1cTSP-1-2 will berandomly selected and used as the pooled populations together with theestablished TA3 and LLC transfectants expressing a similar level ofADAMTS-1CTF or ADAMTS-1NTF, or transfected with the expression vectoralone in the s.c. tumor growth and metastasis experiments.

mTSP-1 domain inhibits growth and metastasis of TA3 and LLC cells in asimilar extent as that of ADAMTS-1_(NTF); while expression of each ofthe cTSP-1 domains display a weaker anti-tumor effect compared to thatcaused by ADAMTS-1_(CTF), which contains two TSP-1 modules. These smallrecombinant proteins (53-56 amino acid long) are used as anti-cancersagents.

Deletions and Mutations in the m or cTSP-1 Domains of ADAMTS-1 andEstablish TA3 and LLC Transfectants Expressing these ADAMTS-1 Mutants

Within in the TSP type I repeats of thrombospondin-1, in addition toWXXWXXW motif, the CSVTCG motif, which binds to CD36, has been shown tocontain anti-tumor and anti-angiogenic activity. The _(m/c)TSP-1 domainsof ADAMTS-1 contain the motifs that are similar to WXXWXXW and/or CSVTCG(SEQ ID NO:47) motifs. In addition, the consensus motif search (GCGgenomics) has demonstrated that the most consensus motif among the m andcTSP-1 domains of ADAMTS-1 is the WGE/DCSKTC (SEQ ID NO:50) motif (FIG.18). Thus, three deletions in _(m/c)TSP-1 domains are made: WGPWGPWGD(ADAMTS-1_(mTSP-1WXXWde1-)SEQ ID NO:48) or WV/QI/VE/GE/DWG/S(ADAMTS-1_(cTSP-1WXXXXWde1)), WGDCSRTC (ADAMTS-1_(mTSP-1WGde1-)SEQ IDNO:49) or WG/SE/PCSKTC (ADAMTS-1_(cTSP-1WG/Sde1-)SEQ ID NO:51), CSRTCGGG(ADAMTS-1_(mTSP-1CSde1-)SEQ ID NO:52) or CSKTCGS/KG(ADAMTS-1_(cTSP-1CSde1-)SEQ ID NO:53, FIG. 18).

The deletional mutagenesis are performed as described usingfADAMTS-1_(mTSP-1) and fADAMTS-1_(cTSP-1-1), or fADAMTS-1_(cTSP-1-2) (inpEF/6/v5-His expression vectors) as the templates. These expressionconstructs are used to transfect Cos-7 cells transiently to assess theexpression capacity of these v5-epitope tagged fragments. All thedeletional mutants are established in cell lines and their properexpression in Cos-7 cells is demonstrated. These deletional constructsare used to transfect TA3_(wt1) and LLC_(wt1) cells. Five independentTA3 or LLC transfectants expressing a high to intermediate level of eachof the mutants are used as the pooled populations in the s.c. tumorgrowth and metastasis experiments together with the established TA3 andLLC transfectants expressing ADAMTS-1_(NTF), ADAMTS-1_(CTF),fADAMTS-1_(mTSP-1) and fADAMTS-1_(cTSP-1-1), or fADAMTS-1_(cTsp-1-2), ortransfected with the expression vector alone (the control).

The disclosures of each and every patent, patent application,publication, and accession number cited herein are hereby incorporatedherein by reference in their entirety. The appended sequence listing ishereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. An isolated polypeptide fragment of ADAMTS-1 thatinhibits tumor growth and/or metastasis, wherein the fragment consistsof SEQ ID Nos: 5, 7, 9, and or
 11. 2. A pharmaceutical compositioncomprising a polypeptide of claim
 1. 3. A composition comprising atleast two different polypeptide fragment of ADAMTS-1 that inhibit cellproliferation or metastasis, wherein said fragment comprises SEQ ID Nos:5, 7, 9, and/or
 11. 4. The position of claim 3 wherein said compositionis a pharmaceutical composition.
 5. An isolated polynucleotide encodinga polypeptide fragment of ADAMTS-1 wherein said fragment inhibits tumorgrowth and/or metastasis, wherein said polynucleotide comprises SEQ IDNO: 6, 8, 10, or
 12. 6. A pharmaceutical composition comprising theisolated polynucleotide of claim
 6. 7. The isolated polynucleotide ofclaim 6 wherein said polynucleotide is a vector or plasmid.
 8. A methodfor identifying an inhibitor or an activator of ADAMTS-1 auto-cleavagecomprising performing a test assay comprising: a) contacting ADAMTS-1with a test compound under conditions in which ADAMTS-1 undergoescleavage in the absence of a test compound; b) measuring cleavage levelof ADAMTS-1; and c) comparing the cleavage level to cleavage level ofADAMTS-1 in the absence of the test compound, wherein a decrease inauto-cleavage indicates that the test compound is a cleavage inhibitoror wherein an increase in auto-cleavage indicates that the test compoundis a cleavage activator.
 9. The method of claim 8 wherein said the testcompound is contacted with a cell comprising ADAMTS-1.
 10. The method ofclaim 9, further comprising performing, a negative control assay whichcomprises contacting a cell that does not comprise ADAMNTS-1 or a cellthat comprises a cleavage resistant mutant of ADAMTS-1.
 11. The methodof claim 9, further comprising performing a positive control assay whichcomprises contacting a cell comprising ADAMTS-1 a positive controlcompound and measuring cleavage.
 12. The method of claim 8, furthercomprising measuring the cleavage of ADAMTS-1 in the absence of the testcompound.
 13. A method for identifying a heparin inhibitor comprising:a) contacting a composition comprising heparin and ADAMTS-1 with a testcompound under conditions in which ADAMTS-1 undergoes auto-cleavageand/or proteolytic cleavage in absence of heparin; b) measuring cleavagelevel of ADAMTS-1; and c) comparing cleavage level of ADAMTS-1 in theabsence of the test compound; wherein an increase in the cleavage ofADAMTS-1 indicates that the compound is a heparin inhibitor.
 14. Amethod of identifying a metalloproteinase inhibitor comprising: a)contacting a ADAMTS-1 polypeptide or fragment thereof comprisingmetalloproteinase activity with a test compound under conditions whichmetalloproteinase activity is detected in the absence of the testcompound. b) measuring metalloproteinase activity level of ADAMTS-1; andc) comparing the metalloproteinase activity level of ADAMTS-1 in thepresence or absence of the test compound, wherein a decrease inmetalloproteinase activity indicates the test compound is ametalloproteinase inhibitor.
 15. The method of claim 14 wherein saidfragment comprises SEQ ID NO: 5, 7, 9, and/or
 11. 16. The method ofclaim 14 wherein the metalloproteinase activity of ADAMTS-1 is comparedto a fragment or mutant of ADAMTS-1 that has no metalloproteinaseactivity.
 17. The method of claim 16 wherein said fragment or mutant ofADAMTS-1 that has no metalloproteinase activity comprises SEQ ID NO 31,33, 35, and/or
 36. 18. A method of treating cancer in an individualcomprising administering to the individual a therapeutically effectiveamount of a polypeptide fragment of ADAMTS-1 and/or a nucleic acid thatencodes a polypeptide fragment of ADAMTS-1 that inhibits cellproliferation and/or metastasis.
 19. The method of claim 18 wherein thepolypeptide fragment comprises a TSP type-I motif.
 20. The method claim18 wherein the fragment comprises SEQ ID NO: 5, 7, 9 and/or
 11. 21. Themethod of claim 18 wherein the nucleic acid molecule encoding thepolypeptide fragment comprises SEQ ID NO: 6, 8, 10, and/or
 11. 22. Themethod of claim 18 wherein said polypeptide fragment of ADAMTS-1comprises the spacer/Cys-rich and/or spacer domain of ADAMTS-1 or anucleic acid molecule encoding a polypeptide fragment of ADAMTS-1comprising the spacer/Cys-rich and/or spacer domain of ADAMTS-1.
 23. Themethod of claim 22 wherein said fragment comprises SEQ ID NO: 21 and/or23.
 24. The method of claim 22 wherein said nucleic acid moleculecomprises SEQ ID NO: 22 and/or
 24. 25. A method of mating cancercomprising administering an inhibitor of the metalloproteinase activityof ADAMTS-1.
 26. The method of claim 25 wherein the inhibitor is ametalloproteinase defective polypeptide of ADAMTS-1 or a nucleic acidmolecule encoding a metalloproteinase defective polypeptide of ADAMTS-1.27. The method of claim 26 wherein the metalloproteinase defectivepolypeptide of comprises SEQ ID NO: 29, 31, 33, and/or
 35. 28. Themethod of claim 26 wherein nucleic acid molecule comprises SEQ ID NO:30, 32, 34, and/or
 36. 29. The method of claim 25 wherein said inhibitoris an antibody that binds to ADAMTS-1.